Hmgb1-derived peptides enhance immune response to antigens

ABSTRACT

The invention provides an immunostimulatory peptide containing the amino acid sequence SAFFLFCSE and uses thereof. 
     The invention also provides an immunostimulatory peptide containing the amino acid sequence DPNAPKRPPSAFFLX 1 X 2 X 3 X 4  or derivatives thereof. In one embodiment, when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.

This invention was made with government support under Grant No. W81XWH-07-1-0412 awarded by U.S. Army Medical Research and Materiel Command and Grant No. 5 U54 CA119335 awarded by National Institutes of Health/National Cancer Institute. The government has certain rights in the invention.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Vaccines traditionally have, and still consist of whole-inactivated or live-attenuated pathogens or toxins [1,2]. The usage of these modified pathogens is, however, unattractive for several reasons. Live attenuated pathogens can cause disease by reverting to a more virulent phenotype, especially in the non-developed immune system of newborns or immunodeficient patients, and whole inactivated pathogens contain reactogenic components that can cause undesirable vaccine side effects. Therefore, there is growing interest and research to develop a new generation of vaccines containing recombinant protein subunits, synthetic peptides, and plasmid DNA [1]. While these new modalities promise to be less toxic, many are poorly immunogenic when administered without an immune-stimulating adjuvant. As adjuvants are a crucial component of the new generation of vaccines, there is a great need for safer and more potent adjuvants [1-3].

The development of the appropriate type of immune response is essential for successful immunization. Robust cell-mediated immunity, which is associated with a Th1 type immune response, is thought to be required for the control of intracellular pathogens [4], viruses [5] as well as cancer [6]. Humoral immunity, characterized by a Th2 type response is useful for vaccination against extracellular pathogens, such as bacteria. By choosing an appropriate adjuvant, the immune response can be selectively modulated to initiate a Th1 or Th2-type [7]. Aluminum salts (alum), which are the only vaccine adjuvants currently approved by the US Food and Drug Administration for use in humans [8,9] are not ideal adjuvants for certain pathogens, since they favor a Th2 response with weak or absent Th1 responses [10-14]. Although neutralizing antibodies from a Th2 response can be protective against many pathogens, the generation of Th1 and cytotoxic T lymphocyte (CTL) responses are important, playing crucial roles in the protection and recovery from viruses, intracellular bacteria, and cancer cells.

Pathogen associated molecular patterns (PAMPs) are small molecular sequences commonly associated with pathogens, such as CpG unmethylated bacterial DNA sequences, lipopolysaccharide (LPS), or poly(I:C) [15-18]. While many PAMPs have been investigated for their use as vaccine adjuvants, their development has been slowed for several reasons, including reactogenicity, toxicity, and ability to induce or exacerbate autoimmune diseases [19]. For instance, CpG oligodeoxynucleotides, which signal through TLR9, can activate antigen-presenting cells, induce a wide variety of cytokines, and generate a potent cellular Th1 immune response in mice, initially showed strong clinical promise [20-23]. However, clinical trials in humans utilizing CpG as a cancer immunotherapy adjuvant failed to produce the potent immune responses that were anticipated, and low TLR9 expression in human plasmacytoid DCs may be implicated [24]. Identification of new adjuvants demonstrating low-toxicity and the ability to stimulate a cellular Th1 response in humans would be a great advancement in the development of vaccines for infectious disease and cancer.

In contrast to PAMPs, endogenous molecules and proteins have been proposed and studied as adjuvants. Examples of such endogenous molecules, or danger-associated molecular patterns (DAMPs), include heat stock proteins, cytokines, and high mobility group box 1 (HMGB1) protein [25,26]. Originally identified as a nuclear protein, HMGB1 modulates the innate immune response when released into the extracellular compartment by necrotic and damaged cells [27,28]. HMGB1 is a potent pro-inflammatory cytokine, released by monocytes and macrophages following exposure to LPS, tumor necrosis factor (TNF)-α or IL-1β and as a result of tissue damage [27,29]. Extracellular HMGB1 promotes the maturation of myeloid and plasmacytoid DCs [30-32] and it has been shown to act as immune adjuvant by enhancing immunogenicity of apoptotic lymphoma cells and eliciting antibody responses to soluble ovalbumin protein [33].

We have previously identified a short peptide, named Hp91, within the B box domain of HMGB1 that induces activation of human and mouse DCs [25]. Hp91-activated DCs show increased secretion of pro-inflammatory cytokines and chemokines, including the Th1 cytokine, IL-12. In addition, DCs exposed to HMGB1-derived peptides induced proliferation of antigen-specific syngeneic T cells in vitro [25]. Here we show novel immunostimulatory peptides act as adjuvants in vivo by enhancing immune responses to peptide and protein antigen.

SUMMARY OF THE INVENTION

The invention provides an immunostimulatory peptide containing the amino acid sequence SAFFLFCSE and uses thereof.

The invention also provides an immunostimulatory peptide containing the amino acid sequence DPNAPKRPPSAFFLX₁X₂X₃X₄ or derivatives thereof. In one embodiment, when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.

In an embodiment of the invention, the derivative is an immunostimulatory peptide having the amino acid sequence RPPSAFFLX₁X₂X₃X₄, wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.

In another embodiment, the derivative is an immunostimulatory peptide having the amino acid sequence SAFFLX₁X₂X₃X₄, wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.

Other embodiments of SAFFLX₁X₂X₃X₄ include those having the amino acid sequence: SAFFLX₁CSE, SAFFLFX₁SE, SAFFLFCX₁E, or SAFFLFCSX₁, wherein X₁ is alanine (A), glycine (G), or valine (V). In a further embodiment, SAFFLX₁X₂X₃X₄ is further mutated so that F at amino acid positions 3 and/or 4 is changed to S.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Hp91 causes release of cytokines in vivo. Mice were injected i.v. into the tail vein with Hp91 (10 or 100 μg) or PBS (denoted as 0 μg Hp91). Blood was collected after 2 and 24 h and serum was analyzed for IFN-γ, IL-6, IL-12 (p70), and TNF-α by ELISA. Data shown are mean (+/−SEM) from 3-4 mice per group.

FIG. 2. Cellular immune response in Hp91 immunized mice. (A-B) Mice were immunized with OVA peptides in PBS (denoted as “0”), Hp91 (250 and 500 μg), or IFA. One group of mice was immunized with OVA protein and Hp91 (500 μg). Freshly-isolated splenocytes from the immunized mice were cultured in the presence of (A) OVA-I (SIINFEKL) peptide (2.5 μg ml⁻¹) or (B) OVA-II (ISQAVHAAHAEINEAGR) (2.5 μg ml⁻¹) in an IFN-γ ELISPOT assay. The number of IFN-γ-secreting cells was determined 18 hours later. The data shown (IFN-γ spot-forming cells per million cells) are means (+/−SEM) for 10 mice/group, except the Hp91-OVA protein group which is n=5. Asterisk, p<0.05 between groups; Student's t-test. Data are representative of at least 3 independent experiments. (C) Freshly isolated splenocytes from PBS/OVA-peptide and Hp91/OVA-peptide immunized mice were depleted of CD8+ or CD4+ T cells and stimulated overnight in the presence of 2.5 μg ml⁻¹ OVA-I (SIINFEKL) peptide in an IFN-γ ELISPOT assay as above. Data shown are means (+/−SEM) for 5 mice per group. (D) Mice were immunized twice with OVA-I (SIINFEKL) peptide (50 ug) co-injected with 0, 30, 60, 125, or 250 μg Hp91 dissolved in PBS. Splenocytes from immunized mice were cultured in an OVA-I IFN-γ ELISPOT assay as above. Spleens were collected 6 days after the boost. Asterisk, p<0.05 between groups; Student's t-test compared to PBS. (E) Mice were immunized with OVA peptides in PBS, Hp91 or Hp121 (250 μg). Mice received an additional boost s.c. into the contralateral flank one month after the first boost. Freshly-isolated or OVA-I-expanded splenocytes were cultured in an OVA-I IFN-γ ELISPOT assay as above. The number of IFN-γ spot-forming cells is shown as means (+/−SEM) for 4 mice per group. Asterisks, (*<0.05 and **<0.005); 2-way ANOVA.

FIG. 3. Cytokine secretion in Hp91 immunized mice. Mice were immunized with OVA peptides (50 μg) co-injected with Hp91 (0, 250 or 500 μg dissolved in PBS) or IFA. One group of mice was immunized with OVA protein and Hp91 (500 μg) in PBS. Splenocytes from immunized mice were stimulated overnight with 2.5 μg ml⁻¹ of (A) OVA-I (SIINFEKL) peptide or (B) OVA-II (ISQAVHAAHAEINEAGR). Culture supernatants were collected and analyzed for IL-2 secretion by ELISA. Data shown are mean (+/−SEM) for 5-10 mice per group. Asterisk, p<0.05; Student's t-test.

FIG. 4. Antibody responses in Hp91 immunized mice. Serum was obtained from immunized mice (5 mice per group) 10 days after the final immunization and analyzed for antibody levels by ELISA. (A) A 1:100 dilution of the serum from immunized mice as indicated, (B) a 1:100 dilution of serum from mice immunized with OVA protein and Hp91 (0 or 250 μg in PBS) or (C) a serial dilution of serum from mice immunized with OVA protein and Hp91 (500 μg) in PBS was added to the plates, followed by a peroxidase-conjugated anti-mouse IgG1 antibody. Plates were developed with TMB substrate and absorbance was analyzed on a microplate reader. Asterisks, p<0.001; Student's t-test.

FIG. 5. CTL Induction in immunized mice. Expanded splenocytes from immunized mice were cultured with E.G7-OVA cells at the indicated effector to target ratios for 6 h. Cell culture supernatants were collected and cytotoxicity was quantified in a CytoTox96 non-radioactive cytotoxicity assay. Data from at least 3 mice per group are shown. Data were analyzed by linear regression and slopes were compared for significance. Asterisk, p<0.05; compared to PBS.

FIG. 6. Hp91/alum adjuvant synergy. Mice were primed and boosted two weeks later with OVA (SIINFEKL) peptide (50 μg) co-injected with PBS, Hp91 (250 μg), Alum (aluminum hydroxide or aluminum phosphate) (500 μg) (Brenntag Biosector A/S), or Hp91 mixed with alum. Splenocytes from immunized mice were stimulated overnight with 2.5 μg ml⁻¹ of OVA (SIINFEKL) peptide in an IFN-γ ELISPOT assay. The number of IFN-γ-secreting cells was determined 18 hours later.

FIG. 7 shows a bar graph of Hp91 fused with antigen peptide HER2 increasing HER2-specific CTL responses in vitro. Immature bone-marrow derived mouse DCs were exposed to media, LPS, or HER2-HP91 fusion peptide for 48 h. DCs were co-cultured with HER2-specific CTLs overnight and the number of IFN-g secreting T cells was evaluated by ELISPOT.

FIG. 8 shows a line graph of tetrameric Hp91 activating dendritic cells in vitro. Immature DCs were exposed to increasing concentrations of free Hp91 and matching amounts of Hp91 tetramer. Cell culture supernatants were collected after 48 h and analyzed for the presence of IL-6 by ELISA.

FIG. 9 shows a bar graph of tetrameric Hp91 activating dendritic cells in vitro through p38 and NF-kB. Immature DCs were pre-treated with the indicated inhibitors or DMSO control for 30 min prior to exposure to tetrameric Hp91. Cell culture supernatants were collected after 48 h and analyzed for the presence of IL-6 by ELISA.

FIG. 10 shows a bar graph of tetrameric Hp91 acting as adjuvant in vivo. Mice were immunized with OVA peptide mixed with PBS, monomeric Hp91 (250 ug), or matching molarity of Hp91 tetramer and boosted two more times. After the final boost, mice were sacrificed and analyzed for OVA-specific CTL responses by ELISPOT.

FIG. 11 shows a line graph of delivery of Hp91 inside of PLGA nanoparticles that leads to increased DC activation in vitro as compared to free Hp91. Immature human DCs were exposed to increasing concentrations of free Hp91 and matching amounts of Hp91 loaded in hybrid PLGA-nanoparticles. Cell culture supernatants were collected after 48 h and analyzed for the presence of IL-6 by ELISA.

FIG. 12. Mice were immunized with OVA peptide mixed with PBS, empty nanoparticles, free Hp91, or matching amounts of Hp91 loaded in hybrid PLGA-nanoparticles and boosted two more times. After the final boost, mice were sacrificed and analyzed for OVA-specific CTL responses by ELISPOT. (A) Interferon-gamma ELISPOT (B) GranzymeB ELISPOT (C) IL-2 secretion from splenocytes. *p<0.05; Student's t-test.

FIG. 13 shows a bar graph and a line graph of Hp91 inducing HER2-specific immune responses in HER2 transgenic mice and increasing the percentage of tumor free mice. Left: HER2neu transgenic mice (n=5) were immunized with HER2 alone or mixed with free Hp91 peptide or Hp91 loaded inside of PLGA-nanoparticles (Hp91) s.c. Mice were boosted twice with the same vaccines. Mice were sacrificed after final boost and immune responses to HER2 were measured by ELISPOT. Right: Mice (n=9-10) were monitored after immunization for tumor growth, the percentage of tumor free mice is depicted over time.

FIG. 14 shows line graphs of intra-tumoral injection of Hp91 peptide free and in nanoparticles leading to tumor (melanoma) growth retardation in vivo. Left: Tumor bearing mice (B16 tumors) received intra tumoral injections of free HP91 (100 ug) or PBS control. Right: Tumor bearing mice (B16 tumors) received intra tumoral injections of Hp91 loaded in PLGA-nanoparticles or matching amounts of empty nanoparticles as control. In both cases the agents were injected daily for 10 days and tumor growth was monitored with a set of calipers over the indicated time period.

FIG. 15 shows bar graphs of Hp91 causing cytokine release from tumor cells. Cell lines were exposed to media control, Hp91 (100 ug/ml), control peptide Hp121 (100 ug/ml), LPS (10 ng/ml), or poly I:C for 1 h, washed and cell culture supernatants were collected after 24 h and analyzed for the indicated cytokines by ELISA.

FIG. 16 shows a bar graph and a line graph of HP-91 packaged into liposome-nanoparticles activates DCs more potently than free Hp91. Left: Immature human DCs were exposed to media, empty liposomes, free Hp91 (100 ug/ml), or different amounts of Hp91 loaded into liposomes for 48 h. Cell culture supernatants were analyzed for the presence of IL-6 by ELISA. Right: Immature human DCs were exposed to media, empty liposomes, free Hp91 (20 ug), 0.5 ug Hp91 loaded into liposomes, or LPS for 48 h. Cells were collected and analyzed for CD83 expression by flow cytometry.

FIG. 17. Hp91 uptake by DCs is dose, time, and sequence dependent (A) Immature human DCs (iDCs) were pre-cooled, incubated on ice for 30 minutes with biotinylated Hp91 or Hp121 (200 μg ml⁻¹) to allow peptide binding, washed, then incubated for 0, 15, or 30 additional minutes at 37° C. Times on figure represent total incubation times (ice and 37° C.). Cells were cytospun, fixed, permeabilized, and stained with Streptavidin-Alexa 488 to visualize biotinylated peptides (Green) and Hoechst DNA stain (Blue). Cells were imaged on a Zeiss LSM confocal microscope. Data are representative of 3 independent experiments. (B) iDCs were pre-cooled on ice for 30 minutes, then incubated with biotinylated-Hp91 (0, 10, or 100 μg ml⁻¹) for 30 minutes at 37° C. Cells were permeabilized with Cytofix/Cytoperm, stained with, and analyzed by flow cytometry. Results shown are representative of 6 independent experiments. (C) iDCs were incubated with biotinylated Hp91 for 10 or 30 minutes (at 100 μg ml⁻¹) and permeabilized, stained, and analyzed as above. Results shown are mean (±SEM) of 4 independent experiments. (D-E) iDCs were pre-cooled on ice for 30 minutes, then incubated with medium only or biotinylated-Hp91, Hp121, or scrambled Hp91 (“Scramble”) at 200 μg ml⁻¹ for 30 minutes at 37° C. Cells were permeabilized, stained, and analyzed by flow cytometry as above. (D) is mean (±SEM) for 3 independent experiments and (E) is a representative result. *p<0.05 compared to medium; Student's t-test. (F-G) Mice were immunized with SIINFEKL peptide in PBS with Hp91 or Scramble. (F) Freshly isolated splenocytes from the immunized mice were cultured in the presence of SIINFEKL peptide (2.5 μg ml⁻¹). The number of IFN-γ-secreting cells was determined 18 h later. The data shown is mean (±SEM) for 5-10 mice/group. *p<0.05 between groups; Student's t-test. (G) Freshly isolated splenocytes from the immunized mice were cultured in the presence of SIINFEKL peptide (2.5 μg ml⁻¹). Supernatants were collected after 18 hours and analyzed for IL-2 secretion by ELISA. The data shown is mean (±SEM) for 5-10 mice/group. *p<0.05 between groups; Student's t-test.

FIG. 18. Hp91 is taken up by clathrin-mediated endocytosis. (A-B) Immature human DCs were pre-cooled on ice for 30 minutes, then incubated with biotinylated Hp91 (200 μg ml⁻¹) for 30 minutes at 4, 16, or 37° C. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. (A) Data are mean (±SEM) of 3 independent experiments and (B) is a representative result. ***p<0.001 compared to 4° C.; Student's t-test. (C) iDCs were pre-treated with the phagocytosis inhibitor Latrunculin B or medium only for 30 minutes before incubation with biotinylated-Hp91 for 30 minutes. Cells were permeabilized, stained, and analyzed by flow cytometry as above. Data are mean (±SEM) of 5 independent experiments. (D) iDCs were pre-treated for 30 minutes with medium only (Med), DMSO control (D), the clathrin-mediated endocytosis inhibitors 1) phenylarsine oxide (PAO) at 2 μM or 2) chlorpromazine (CP) at 100 μM, the energy-dependent endocytosis inhibitor sodium azide (SA) at 10 mM, the caveolin-mediated endocytosis inhibitor nystatin (NY) at 20 μM, or the micropinocytosis inhibitor amiloride (AM) at 2 mM before incubation with biotinylated Hp91 (200 μg ml⁻¹) for 30 minutes. Cells were permeabilized, stained, and analyzed by flow cytometry as above. Results are mean (±SEM) of 3 independent experiments. **p<0.01 compared to DMSO control; Student's t-test.

FIG. 19. p38 MAPK and NFkB are necessary for Hp91-mediated IL-6 secretion from human dendritic cells. Immature human DCs were pre-treated for 30 minutes with DMSO control (D), SB203580 at 5 (S5) or 20 (S20) μM, PD98059 (PD) at 20 μM, or NFkB at 20 μM prior to exposure to Hp91. Cell culture supernatants were collected after 48 h and analyzed for the presence of IL-6 by ELISA. Results are mean (±SEM) for 5 independent experiments. *p<0.05; Student's t-test.

FIG. 20. TLR4, MyD88, and MyD88-dependent and -independent pathways are necessary for Hp91-mediated activation of mouse cells. (A) Immature BM-DCs from wild type (WT) or knockout mice (as indicated) were incubated with Hp91 (200 μg ml⁻¹). Supernatants were collected after 24 hours and analyzed for the presence of IL-6 by ELISA. Results are mean (±SEM) for at least 3 independent experiments. *p<0.05 compared to WT; Student's t-test. (B) J774 macrophages were stimulated for 20, 40, or 60 minutes with Hp91 (200 μg ml⁻¹) or LPS (10 ng ml⁻¹) or left unstimulated (M). Cells were harvested, lysed, and analyzed for p-p38. Immunoblots were probed with anti-p-38, total p38, and GAPDH antibodies. Data shown is representative from 4 separate immunoblots. Non-relevant wells were removed from the blot. (C) J774 macrophages were pre-treated for 30 minutes with DMSO control (D), SB203580 at 20 (SB) μM, PD98059 (PD) at 20 μM, TPCK at 20 μM, Dynasore (DYN) at 80 μM, phenylarsine oxide (PAO) at 2 μM, or chlorpromazine (CP) at 100 μM prior to exposure to Hp91. Cell culture supernatants were collected after 24 h and analyzed for the presence of IL-6 by ELISA. Data shown are mean (±SEM) for triplicates. SB, PD, and TPCK results are representative of 3 independent experiments. *p<0.05; Student's t-test. (D) J774 macrophages were pre-treated for 30 minutes with medium only (Med), DMSO control (D), the dynamin-mediated endocytosis inhibitor Dynasore (DYN) at 80 μM, or the clathrin-mediated endocytosis inhibitors 1) phenylarsine oxide (PAO) at 2 μM or 2) chlorpromazine (CP) at 100 μM before incubation with Cp488-labeled Hp91 (200 μg ml⁻¹) for 30 minutes. Cells were washed and immediately analyzed by flow cytometry as above. Results are mean (±SEM) of 4 independent experiments. *p<0.05 compared to DMSO control; Student's t-test. (E) RAW 264.7 macrophages were pre-treated for 30 minutes with medium only, DMSO control (DMSO), or Dynasore (DYN) at 80 μM prior to stimulation for 20, 40, or 60 minutes with Hp91 (200 μg ml⁻¹) or LPS (10 ng ml⁻¹) or medium only (media). Cells were harvested, lysed, and analyzed for p-IRF3. Immunoblots were probed with anti-p-IRF3, total IRF3, and β-actin antibodies.

FIG. 21. A proposed mechanism of Hp91 signaling.

FIG. 22. HPLC and DC uptake of Hp91 spontaneous dimers. (A-B) Hp91 peptide was dissolved in PBS and incubated at RT in the presence of oxygen for up to 96 h. Peptides were analyzed by HPLC. Peptide monomers show a peak at an earlier time point than the peptide dimers (12.2 min vs. 12.7 min). Percent dimer was determined by measuring the AUC and calculating the dimer AUC/total AUC. Results shown are (A) representative of several independent experiments and (B) mean (±SEM) for 3-4 samples. (C) Hp91 was allowed to form spontaneous air dimers for 24 h prior to reduction with 10 mM DTT for 30 min. Peptides were analyzed by HPLC. (D) Biotinylated Hp91 was incubated for 30 min+/−dithiothreitol and/or H₂0₂. Immature human DCs were incubated with the modified peptides for 30 minutes at 37° C. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. The data shown is mean (±SEM) for 4 independent experiments.

FIG. 23. HPLC and DC activity of Hp91 maleimide dimers. (A) Hp91 peptide control, NEM capped monomer reaction product, or BM(PEG)₂ cross-linked reaction product were analyzed by HPLC. Percent dimer was determined by measuring the AUC and calculating the dimer AUC/total AUC. (B) Immature human DCs were pre-cooled on ice for 30 minutes, then incubated with media only, biotinylated-Hp91 (“Control”) or the biotinylated Hp91 NEM monomer reaction product (“Capped”) (100 mg ml⁻¹) for 60 minutes at 37° C. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. Results shown are mean (±SEM) for 3 independent experiments. (C) Immature DCs were incubated with media only, un-reacted acetylated Hp91 (“Control”), or an acetylated Hp91 NEM monomer reaction product (“Capped”) (100 mg ml⁻¹). Supernatants were collected after 48 hours and analyzed for the presence of IL-6 by ELISA. Data are normalized with respect to media controls. Results are mean (±SEM) for 3 independent experiments. (D) Immature human DCs were pre-cooled on ice for 30 minutes, then incubated with media only, biotinylated-Hp91 (“Control”) or a biotinylated Hp91 BM(PEG)₂ cross-linked reaction product (“Dimer”) (100 mg ml⁻¹) for 60 minutes at 37° C. Cells were permeabilized and stained as above and analyzed by flow cytometry. Results shown are mean (±SEM) for 3 independent experiments. *p<0.05; Student's t-test. (E) Immature DCs were incubated with media only, un-reacted acetylated Hp91 (“Control”), or an acetylated Hp91 BM(PEG)₂ cross-linked reaction product (“Dimer”) (56 mg ml⁻¹). Supernatants were collected after 48 hours and analyzed for the presence of IL-6 by ELISA. Data are normalized with respect to media controls. Results are mean (±SEM) for 3 independent experiments.

FIG. 24. Immunostimulatory activity of Hp91 short peptides. (A) Full length Hp91 (18 aa) is broken into 5 smaller, truncated peptides (8-12 aa). (B) Immature DCs were stimulated with the indicated peptides for 48 hours. Supernatants were analyzed for the presence of IL-6 by ELISA. Data are mean (SEM) for 3 independent experiments. (C, D) Immature DCs were precooled on ice for 30 minutes, then incubated with medium only, Hp91, or truncated Hp91 short peptides (90 μM) for 30 minutes at 37° C. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. (C) is mean (±SEM) of 4 independent experiments and (D) is a representative result. (E-F) Mice were immunized with SIINFEKL peptide in PBS with PBS (negative), UC714, UC1018, or Hp91. Freshly isolated splenocytes from the immunized mice were cultured in the presence of SIINFEKL peptide (2.5 μg ml⁻¹) in an (E) IFN-γ ELISPOT assay, wherein the number of IFN-γ-secreting cells was determined 18 h later, or (F) culture supernatants were collected and analyzed for IL-2 secretion by ELISA. The data shown is mean (±SEM) for at least 5 mice/group. *p<0.05 compared to PBS; Student's t-test. (G-H) Mice were immunized s.c. with apoptotic mitomycin-C treated B16 cells co-injected with PBS (negative) or UC1018 peptide (142 μg) and boosted twice. One week post boost, mice were inoculated s.c. on the flank with 5×10⁵ live B16 cells. (G) Tumor dimensions were measured using calipers and the tumor volume calculated using the following formula; volume=4/3π(a²×b). The data shown is mean (±SEM) for at least 14 mice/group. *p<0.01 compared to PBS; Student's t-test at each time point. (H) Mice were euthanized when tumor volume reached 1.5 cm³. Tumor survival curves were generated, wherein the day of euthanasia was considered as death. ***p<0.001 compared to PBS; Student's t-test.

FIG. 25. Circular dichoism suggests alpha helical shape of UC1018. Hp91, UC18, and UC1018 were dissolved in 75%/25% Trifluoroethanol (TEE, Sigma)/H₂0 (vol/vol) at 200 μg ml⁻¹ and CD spectra were collected on an AVIV Circular Dichroism Spectrometer using a 1 mm pathlength quartz cuvette. These spectra were corrected by subtraction of a “solvent-only” spectrum and smoothed. The spectra are shown in mean residue ellipticity (θ_(mrw)). The Hp91 curve is representative of two independent experiments.

FIG. 26. Immunostimulatory activity of UC1018 mutated peptides. Immature DCs were stimulated with the indicated peptides for 48 hours. Supernatants were analyzed for the presence of IL-6 by ELISA. Data are mean (±SEM) for 4 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION Definitions

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

As used herein, a “derivative” is a molecule that shares sequence similarity and activity of its parent molecule, e.g. DPNAPKRPPSAFFLX₁X₂X₃X₄ or SAFFLX₁X₂X₃X₄. A fragment may be considered a derivative. Also, the derivative may include an immunostimulatory peptide of the invention as part of a larger protein or polypeptide (e.g., a heterologous protein (in one example, the protein is not a HMBG1 protein)). A derivative may include any change to the amino acid sequence and/or chemical quality of the amino acid e.g., amino acid analogs. For example, a derivative of DPNAPKRPPSAFFLX₁X₂X₃X₄ or SAFFLX₁X₂X₃X₄ may include a molecule having an amino acid sequence at least 70% similar to DPNAPKRPPSAFFLX₁X₂X₃X₄ or SAFFLX₁X₂X₃X₄, and/or increases the immunogenicity of any antigen admixed therewith or joined thereto and/or activates the adaptive and/or innate immune system (e.g., induces the costimulatory signal necessary for activation of lymphocytes). The derivative may also maintain an alpha helix conformation.

As used herein, “administer” or “administering” to a subject includes but is not limited to tumoral administration, intratumoral administration, peritumoral administration, intravenous (i.v.) administration, intraperitoneal (i.p.) administration, intramuscular (i.m.) administration, subcutaneous administration, oral administration, inhalation administration, topical administration, administration by injection, as a suppository, or the implantation of a slow-release device such as a miniosmotic pump, or administration by slow release devices such as vesicles or capsules to the subject.

As used herein, “subject” means any living organism to which the agents can be administered in order to regulate an immune response. Subjects may include, but are not limited to, humans, monkeys, cows, goats, sheep, mice, rats, cats, dogs, hamsters, and any transgenic animals.

As used herein, “pharmaceutically acceptable carrier” means any material that may be combined with an immunostimulatory peptide(s) of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in order to administer them to a subject in any form. For example, a carrier includes any material that will maintain the agents' effective activity when administered to a subject and that is non-reactive with a subject's immune system. Potential carriers may include, but are not limited to, any solvents, media, suspensions, emulsions or other excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acids, stearate salts, talcum, oils, gums, glycols, flavorings, preservatives or color additives, etc. Potential carrier forms may include sterile solutions, aerosols, liposomes, vesicles, suppositories, pills, tablets or capsules.

In order that the invention herein described may be more fully understood the following description is set forth.

COMPOSITIONS OF THE INVENTION

The invention provides an immunostimulatory peptide containing the amino acid sequence SAFFLFCSE. In another embodiment, the immunostimulatory peptide consists of the amino acid sequence SAFFLFCSE.

The invention also provides an immunostimulatory peptide containing the amino acid sequence DPNAPKRPPSAFFLX₁X₂X₃X₄ or derivatives thereof. In one embodiment, when X1 is any of the amino acid alanine (A), glycine (G), or valine (V) then X2 is cysteine (C), X3 is serine (S) and X4 is glutamic acid (E); wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is phenylalanine (F), X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S. In a further embodiment, X1 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L) and X2 is cysteine (C), X3 is serine (S) and X4 is glutamic acid (E). In another embodiment, X1 is phenylalanine (F); X2 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L); X3 is S and X4 is E. In a yet further embodiment, X1 is phenylalanine (F); X2 is cysteine (C); X3 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L); and X4 is E. In a still further embodiment, X1 is F; X2 is C; X3 is S; and X4 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L). A list of the amino acids and their one letter symbol is found supra.

In an embodiment of the invention, the derivative may be an immunostimulatory peptide having the amino acid sequence RPPSAFFLX₁X₂X₃X₄, wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.

In another embodiment, the derivative may be an immunostimulatory peptide having the amino acid sequence SAFFLX₁X₂X₃X₄. In an embodiment of SAFFLX₁X₂X₃X₄, when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S. In another embodiment, X1 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L) and X2 is cysteine (C), X3 is serine (S) and X4 is glutamic acid (E). In another embodiment, X1 is phenylalanine (F); X2 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L); X3 is S and X4 is E. In a further embodiment, X1 is phenylalanine (F); X2 is cysteine (C); X3 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L); and X4 is E. In a still further embodiment, X1 is F; X2 is C; X3 is S; and X4 may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L). In a yet further embodiment, SAFFLX₁X₂X₃X₄ may be further mutated so that F at amino acid positions 3 and/or 4 is changed to S.

Other embodiments of SAFFLX₁X₂X₃X₄ include those having the amino acid sequence: SAFFLX₁CSE, SAFFLFX₁SE, SAFFLFCX₁E, or SAFFLFCSX₁, wherein X₁ is alanine (A), glycine (G), or valine (V).

The immunostimulatory peptides of the invention (or its derivatives) may be monomeric, dimeric, trimeric, or tetrameric. Particular dimeric embodiments include, but are not limited to, dimeric SAFFLFCSE, dimeric DPNAPKRPPSAFFLX₁X₂X₃X₄, dimeric RPPSAFFLX₁X₂X₃X₄, and dimeric SAFFLX₁X₂X₃X₄. Dimeric peptides of the invention may be homodimeric or heterodimeric.

The immunostimulatory peptides of the invention (or its derivatives) may be soluble (i.e., circulating) or bound to a cell surface. The immunostimulatory peptides of the invention (or its derivatives) can be made synthetically or recombinantly.

The immunostimulatory peptide (or its derivatives) may also exhibit an alpha helix conformation. In one embodiment, the alpha helix conformation may provide a circular dichroism profile that shows negative peaks at about 205-207 nm and/or 222 nm. Further, the alpha helix conformation may provide a circular dichroism profile that shows a positive peak below about 200 nm.

In accordance with the practice of the invention, SAFFLX₁X₂X₃X₄ molecules may include a non-SAFFLX₁X₂X₃X₄ attached or joined thereto. For example, additional peptide sequences may be added or joined to the N-terminal or C-terminal end of SAFFLX₁X₂X₃X₄. In one embodiment, the immunostimulatory peptide SAFFLX₁X₂X₃X₄ further comprises on its N-terminal portion the amino acid sequence arginine (R), proline (P), and proline (P). This immunostimulatory peptide has the sequence RPPSAFFLX₁X₂X₃X₄. X₁, X₂, X₃ and X₄ may be any of the amino acids alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L) as described above.

In another embodiment, the immunostimulatory peptide DPNAPKRPPSAFFLX₁X₂X₃X₄ may be further mutated so that F at amino acid positions 12 and/or 13 is changed to S.

Specific examples of the immunostimulatory peptide, SAFFLX₁X₂X₃X₄, include, but are not limited to SAFFLX₁CSE, SAFFLFX₁SE, SAFFLFCX₁E, or SAFFLFCSX₁, wherein X₁ may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L). Preferred examples include SAFFLACSE and SAFFLFASE.

Specific examples of the immunostimulatory peptide, RPPSAFFLX₁X₂X₃X₄, include, but are not limited to RPPSAFFLX₁CSE, RPPSAFFLFX₁SE, RPPSAFFLFCX₁E, or RPPSAFFLFCSX₁, wherein X₁ may be alanine (A), glycine (G), valine (V), isoleucine (I), or leucine (L). Preferred examples include RPPSAFFLACSE and RPPSAFFLFASE.

In a further embodiment, the immunostimulatory peptide RPPSAFFLX₁X₂X₃X₄ is further mutated so that F at amino acid positions 6 and/or 7 is changed to S.

In accordance with the practice of this invention, the peptides of the invention may have additional amino acid substitutions in the amino acid sequence especially, for example, corresponding to the X₁ X₂ X₃ X₄ domain (of e.g. DPNAPKRPPSAFFLX₁X₂X₃X₄ or SAFFLX₁X₂X₃X₄) so as to produce molecules which would retain the functional property of DPNAPKRPPSAFFLX₁X₂X₃X₄ or SAFFLX₁X₂X₃X₄, namely, the molecule having such substitutions will still maintain an alpha helix conformation and/or increase the immunogenicity of any antigen admixed therewith or joined thereto and/or activates the adaptive and/or innate immune system (e.g., induces the costimulatory signal necessary for activation of lymphocytes. The mutation(s) may include one or more amino acid residues substituted with an amino acid having conservative (e.g., substitute a leucine with an isoleucine) or non-conservative (e.g., substitute a glycine with a tryptophan) structure or chemical properties, amino acid deletions, additions, frameshifts, or truncations.

For example, it is a well-established principle of protein chemistry that certain amino acid substitutions, entitled “conservative amino acid substitutions,” can frequently be made in a protein without altering either the conformation or the function of the protein. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine and valine (V).

Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments.

A mutation in DNA may change a codon leading to a change in the amino acid sequence. A DNA change may include substitutions, deletions, insertions, alternative splicing, or truncations. An amino acid change may include substitutions, deletions, insertions, additions, truncations, or processing or cleavage errors of the protein. Alternatively, mutations in a nucleotide sequence may result in a silent mutation in the amino acid sequence as is well understood in the art. In that regard, certain nucleotide codons encode the same amino acid. Examples include nucleotide codons CGU, CGG, CGC, and CGA encoding the amino acid, arginine (R); or codons GAU, and GAC encoding the amino acid, aspartic acid (D). Thus, a protein can be encoded by one or more nucleic acid molecules that differ in their specific nucleotide sequence, but still encode protein molecules having identical sequences. The amino acid coding sequence is as follows:

One Amino Acid Symbol Symbol Letter Codons Alanine Ala A GCU, GCC, GCA, GCG Cysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic Acid Glu E GAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA, GGG Histidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys K AAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUG Asparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine Gln Q CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU, UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val V GUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

The peptides of the invention may comprise amino acids that are naturally occurring or non-naturally occurring. The natural amino acid in the wild-type polypeptide may be replaced with a desired non-natural amino acid.

As used herein, a “natural amino acid” is the 20 naturally occurring amino acids, namely glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine and proline.

As used herein, the term “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and/or reactivity of a natural amino acid. Thus, all amino acids but the natural 20 amino acids are referred to as non-natural amino acids even if they are naturally present (e.g. hydroxyproline).

The immunostimulatory peptides of the invention (or derivatives thereof) may be joined to a tag or crosslinking agent (e.g. a non-immunogenic tag or crosslinking agent). Merely by way of example, the tag or crosslinking agent may be any of polyalkylene glycol (PAG)(e.g., linear or branched polyalkylene glycol) and a lipid. The polyalkylene glycol may be a polyethylene glycol (PEG).

The invention also provides compositions comprising the immunostimulatory peptides of the invention (or derivatives thereof) combined with nanoparticles or microparticles and/or a pharmaceutically acceptable carrier.

The invention further provides vaccine formulations comprising an antigen (e.g. a heterologous antigen) and an immunostimulatory peptide of the invention (or a derivative thereof). The antigen may be admixed with the immunostimulatory peptide of the invention or joined thereto. The vaccine may include combinations of the immunostimulatory peptides of the invention. The vaccine induces an immune response to the specific antigen admixed therewith or joined thereto. A heterologous antigen is an antigen that is not normally or naturally found adjacent to the immunostimulatory peptides of the invention or its parent molecule.

Suitable examples of antigens include but are not limited to respiratory disease antigens, tumor related antigens, allergy related antigens, and cardiovascular disease antigens. In other embodiments, the antigen is a non-viral environmental irritant. In one embodiment, the antigen is an allergen. In other embodiments, the antigen may be from a bacteria, fungus, mold, dust mite, animal dander or pollen antigen, or a combination thereof. As used herein, antigen includes derivatives or portions of antigens that are immunogenic.

In one embodiment, the respiratory disease antigen may be a respiratory virus antigen. The viral antigens may be inactivated or attenuated. Examples of suitable respiratory viruses include but are not limited to rhinoviruses, influenza viruses, coronavirus RSV, or any combination thereof. Examples of influenza viruses include H1N1, H1N2, H1N7, H2N2, H3N1, H3N2, H3N8, H4N8, H5N1 H5N2, H5N3, H5N8, H5N9, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, and any other subtypes arising from re-assortment between influenza A viruses. Additional examples of respiratory viruses include a coronavirus (e.g., SARS virus), RSV, or a combination thereof.

Specific examples of allergen-related protein antigens useful in the methods and compositions of the present invention include, but are not limited to: allergens derived from pollen, such as those derived from trees such as Japanese cedar (Cryptomeria, Cryptomeria japonica), grasses (Gramineae), such as orchard-grass (Dactylis, Daetylis glomerata), weeds such as ragweed (Ambrosia, Ambrosia artemisiffiblia); specific examples of pollen allergens including the Japanese cedar pollen allergens (J Allergy Clin Immunol. (1983) 71: 77-86) and (FEBS Letters (1988) 239: 329-332), and the ragweed allergens Amb a 1.1, Amba 1.2, Amb a 1.3, Amb a 1,4, Amb a I1 etc.; allergens derived from fungi (Aspergillus, Candida, 41temaria, etc.); allergens derived from mites (allergens from Dermatophagoidespteronyssinus, Derinatophagoidesfarinae etc.; specific examples of mite allergens including Der p I, Der p II, Der p III, Der p VII, Der f I, Der f II, Der f III, Der f VII etc.); house dust; allergens derived from animal skin debris, feces and hair (for example, the feline allergen Fel d 1); allergens derived from insects (such as scaly hair or scale of moths, butterflies, Chironomidae etc., poisons of the Vespidae, such as Vespa maizdarinia); food allergens (eggs, milk, meat, seafood, beans, cereals, fruits, nuts and vegetables etc.); allergens derived from parasites (such as roundworm and nematodes, for example, Anisakis); and protein or peptide based drugs (such as insulin). Many of these allergens are commercially available.

In certain embodiments, the vaccine formulation (or composition (e.g., comprising the antigen and the immunostimulatory peptide of the invention, together or separate)) may be formulated for use in a child or adult subject.

The vaccine formulation of the invention may further comprise nanoparticles or microparticles. The nanoparticles may be hybrid PLGA-nanoparticles, vesosomes or liposome nanoparticles.

The vaccine formulation may further include an adjuvant, which includes but is not limited to, Alum (e.g., aluminum hydroxide or aluminum phosphate) (Brenntag Biosector A/S), CpG oligodeoxynucleotides (or CpG ODN) (Integrated DNA Technologies), polyinosinic-polycytidylic acid [poly(I:C)] (Invivogen), 2,6,10,15,19,23-Hexamethyltetracosa-2,6,10,14,18,22-hexaene (squalene), monophosphoryl lipid A (MPL), CD40L, (e.g. gp39 or anti-cd40 ab), and/or IL-2.

The invention additionally provides adjuvants comprising the immunostimulatory peptide of the invention (or derivatives thereof) so as to increase the immunogenicity of an antigen admixed therewith or joined thereto. The adjuvant may further comprise nanoparticles or microparticles. In accordance with the practice of the invention, the nanoparticles may be hybrid PLGA-nanoparticles, vesosomes or liposome nanoparticles.

METHODS OF THE INVENTION

Additionally, the invention further provides methods for inducing cytotoxic T lymphocytes (CTLs). In one embodiment, the method comprises contacting or subjecting T cells with an immunostimulatory peptide(s) of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in an amount sufficient to induce CTL proliferation or growth.

The invention also provides methods for enhancing an immune response by administering to the subject an immunostimulatory peptides of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in an amount sufficient to enhance an immune response in the subject.

In accordance with the practice of the invention, the methods may be an ex vivo method, an in vivo method, or an in vitro method.

The invention also provides methods of enhancing an immune response in a subject. In one embodiment, the method comprises administering a sufficient amount of the immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) thereby enhancing the immune response in the subject.

In one embodiment, the invention provides methods for selectively eliciting a cell-mediated immune response but not a humoral immune response in a subject. In one embodiment, the method comprises administering a sufficient amount of the vaccine formulation of the invention thereby enhancing the cell-mediated immune response in the subject.

In another embodiment, the invention provides methods for selectively eliciting a cell-mediated immune response and a humoral immune response in a subject. In one embodiment, the method comprises administering a sufficient amount of the vaccine formulation of the invention thereby enhancing the cell-mediated and humoral immune response in the subject.

The invention further provides methods of inhibiting tumor growth associated with a cancerous disease. In one embodiment, the method comprises administering to a subject an immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in an amount sufficient to inhibit tumor growth in the subject.

In another embodiment, the invention provides methods for reducing the tumor size of a tumor associated with a cancerous disease. The method provides administering to a subject an immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in an amount sufficient to reduce the tumor size in a subject.

In a further embodiment, the invention provides methods for delaying the occurrence of a cancerous disease in a subject. The method comprises administering to the subject an immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) in a dose effective for delaying the occurrence of the cancerous disease.

In another embodiment, an effective amount of an alkylating agent is further administered to the subject prior to, concurrently, or after administering the immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them). The alkylating agent may be a cyclophosphamide. Other alkylating agents may be used.

In an embodiment of the invention, an effective amount of the cyclophosphamide may be between 1 mg/m² and 500 mg/m². Other doses are possible. A further discussion is provided supra.

Examples of cancerous diseases include but are not limited to prostate cancer, prostate cancer metastasized to bone, ovarian cancer, tonsil cancer, bladder cancer, stomach cancer, kidney cancer, testicular cancer, small intestinal cancer, colon cancer, colorectal cancer, cervical cancer, uterine cancer, salivary cancer, throat cancer, bronchial cancer, head and neck cancer, adrenal cancer, neuroblastoma, pheochromocytoma, liver cancer, lung cancer, thyroid cancer, breast cancer, melanoma, schwannoma, neurofibrosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, leiomyosarcoma, leukemia, lymphoma, and pancreatic cancer.

Administration of the immunostimulatory peptide of the invention (or derivatives thereof) (and/or compositions/vaccines/adjuvants including them) may be effected by a delivery system which includes, but is not limited to, an implantable pump, continuous infusion, gene therapy, lipososomes, and injection. Additionally, administration may be effect by various administration means, e.g., oral administration, intratumoral administration, peritumoral administration, intravenous administration, intrarectal administration, intravaginal administration, intrabronchial administration, topical administration, intramuscular administration, intraperitoneal administration, intrapleural administration, by inhalation, by eye drops or by any enteral, parenteral or non-parenteral mechanism or means.

In accordance with the practice of the invention, the subject includes, but is not limited to, human, non-human primates, cows, pigs, horses, goats, sheep, rabbits, pigs, dogs, cats, mice, or rats.

Dosages

The appropriate dosage of immunostimulatory peptides of the invention (or derivatives thereof) (or compositions/vaccines/adjuvants including them) for use in accordance with the methods of the present invention may depend on a variety of factors. Such factors may include, but are in no way limited to, a subject's physical characteristics (e.g., age, weight, sex), whether the compound is being used as single agent or adjuvant therapy, the type of MHC restriction of the patient, the progression (i.e., pathological state) of the infection, and other factors that may be recognized by one skilled in the art.

One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of immunostimulatory peptides of the invention (or derivatives thereof) (or compositions/vaccines/adjuvants including them) which would be required to treat the subject. Generally, an effective dosage may be in the range of about 0.0001 mg to about 1000 mg per kg body weight per 24 hours.

Typically, in therapeutic applications, the treatment would be for the duration of the disease state or condition. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques. It will also be apparent to one of ordinary skill in the art that the optimal course of treatment can be ascertained using conventional course of treatment determination tests.

Where two or more therapeutic entities are administered to a subject “in conjunction”, they may be administered in a single composition at the same time, or in separate compositions at the same time or in separate compositions separated in time. In certain embodiments, the methods of the invention involve the administration of immunostimulatory peptides of the invention (or derivatives thereof) (or compositions/vaccines/adjuvants including them)) in multiple separate doses. Accordingly, the methods for the prevention (i.e. vaccination) and treatment described herein encompass the administration of multiple separated doses to a subject, for example, over a defined period of time. Accordingly, the methods for the prevention (i.e. vaccination) and treatment disclosed herein include administering a priming dose of immunostimulatory peptides of the invention (or derivatives thereof) (or compositions/vaccines/adjuvants including them). The priming dose may be followed by a booster dose. The booster may be for the purpose of revaccination. In various embodiments, the composition or vaccine is administered at least once, twice, three times or more.

KITS

According to another aspect of the invention, kits are provided. Kits according to the invention include package(s) or containers comprising an immunostimulatory peptides of the invention (or derivatives thereof) (or compositions/vaccines/adjuvants including them). The kit may further include an instruction letter for the treatment and/or prophylaxis of a disease, for example, a veterinary disease.

The phrase “package” means any vessel containing compounds or compositions presented herein. In preferred embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

The kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.

Kits may optionally contain instructions for administering compounds or compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of compounds herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compounds. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compounds in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

The kit may optionally also contain one or more other compounds for use in combination therapies as described herein. In certain embodiments, the package(s) is a container for intravenous administration. In other embodiments, compounds are provided in an inhaler. In still other embodiments compounds are provided in a polymeric matrix or in the form of a liposome.

The following examples are provided to further illustrate aspects of the invention. These examples are non-limiting and should not be construed as limiting any aspect of the invention.

EXAMPLES Example 1 Materials and Methods Reagents and Cell Lines

The OVA-transfected EL4 line, E.G7-OVA (ATCC, Manassas, Va., USA), was cultured in RPMI 1640 medium (Invitrogen, Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, Calif., USA), 10 mM HEPES (Invitrogen), and penicillin (100 U ml⁻¹)-streptomycin (100 μg ml⁻¹)-L-glutamine (2 mM) (Invitrogen).

Peptides and Protein

The peptides, including the ISP Hp91 (DPNAPKRPPSAFFLFCSE), Hp121 (SIGDVAKKLGEMWNNTAA), the MHC-Class I (H-2K^(b))-restricted peptide epitope of ovalbumin (OVA-I: OVA 257-264 aa, SIINFEKL), and the MHC-Class II (I-A^(b))-restricted peptide epitope of ovalbumin (OVA-II: OVA 323-339 aa, ISQAVHAAHAEINEAGR) were all purchased from GenScript Corp (Piscataway, N.J., USA) and CPC Scientific (San Jose, Calif., USA). Hp91 and Hp121 peptides were synthesized with an N-terminal biotin. Peptides were routinely synthesized with greater than 95% purity. LPS-free chicken egg white ovalbumin protein was kindly provided by Dr. Thomas Moran (Department of Microbiology, Mount Sinai School of Medicine, New York, N.Y., USA). Unless otherwise stated, all peptides and proteins were dissolved in PBS in preparation for immunization.

Mice and Immunizations

Female C57BL/6 mice 8-12 weeks of age were used for most experiments. All mice were purchased from Charles River Laboratories (Boston, Mass., USA) and housed at the Moores UCSD Cancer Center animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee of UCSD and were performed in accordance with the institutional guidelines. For most experiments, mice were immunized s.c. with 50 μg of SIINFEKL (OVA-I) peptide and 50 μg of ISQAVHAAHAEINEAGR (OVA-II) peptide. The OVA peptide was co-administered with either Hp91 (30 to 500 μg) or PBS (negative control). For some experiments, a protein vaccine group was included, wherein 500 μg of Hp91 was co-administered with 100 μg of LPS-free OVA protein. As a positive control, mice were immunized s.c. with OVA peptide(s) or protein in Incomplete Freund's Adjuvant “IFA” (Sigma-Aldrich, St. Louis, Mo., USA). If not otherwise indicated, mice were immunized and boosted two weeks later and spleens and blood were collected 10-14 days after the final immunization.

Intravenous Administration of ISP

C57BL/6 mice were injected i.v. with 0, 10 or 100 μg Hp91 dissolved in PBS into the tail vein. Blood was collected after 2 h and 24 h by retroorbital puncture. Blood was allowed to clot and serum was isolated after centrifugation. Serum was diluted and analyzed for systemic cytokine and chemokine release by ELISA (eBioscience, San Diego, Calif., USA).

Detection of Antigen-Specific Antibody Production by ELISA

Serum was obtained by retroorbital puncture or cardiac puncture from mice following immunization. Blood was allowed to clot and serum was isolated after centrifugation. Microtiter plates were coated overnight with OVA protein (Sigma-Aldrich), blocked with BSA, and dilutions of serum were added to the plates for incubation. Plates were washed, incubated with anti-mouse IgG or IgM peroxidase conjugated antibodies (Roche, Basel, Switzerland), developed using Zymed TMB substrate (Invitrogen), and analyzed using a microplate reader at 450 nm.

Spleen Cell Preparation

Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70 μm nylon cell strainer (BD Biosciences, Franklin Lakes, N.J., USA). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, Ind., USA) and the splenocytes were subsequently resuspended in complete medium (RPMI 1640 with 10% FBS, L-glutamine, penicillin, streptomycin, and HEPES).

In some experiments, CD4+ and CD8+ cells were depleted from bulk splenocyte populations using anti-CD4 or anti-CD8a conjugated microbeads (Miltenyi-Biotec, Auburn, Calif., USA) according to the manufacturer's instructions.

Enzyme-Linked Immunospot Assay

Freshly isolated splenocytes were plated in duplicate to wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (Millipore, Millerica, Mass., USA) that had been previously coated overnight with 5 μg ml⁻¹ monoclonal anti-mouse IFN-γ antibody (Mabtech, Stockholm, Sweden). Splenocytes were cultured overnight at 37° C. with 2.5 μg ml⁻¹ SIINFEKL (OVA-I) peptide, 2.5 μg ml⁻¹ ISQAVHAAHAEINEAGR (OVA-II) peptide, or left unstimulated (medium only). After 18 h, culture supernatants were collected for cytokine analysis and ELISPOT plates were developed using 1 μg ml⁻¹ biotinylated anti-mouse IFN-γ antibody (Mabtech), Streptavidin-HRP (Mabtech), and TMB Substrate (Mabtech). The plate was scanned and the spots were counted using an automated ELISpot Reader System (CTL ImmunoSpot, Shaker Heights, Ohio, USA).

Measurement of Cytokines and Chemokines

Splenocytes were cultured overnight with 2.5 μg ml⁻¹ OVA-I peptide, 2.5 μg ml⁻¹ OVA-II peptide, 5 μg ml⁻¹ concanavalin A positive control (Sigma), or left unstimulated (media only). After 18 h, cell culture supernatants were collected and analyzed for the presence of IL-2 and IL-4 by ELISA (eBioscience).

LDH Cytotoxicity Assay

Splenocytes were expanded in culture at 3×10⁶ cells ml⁻¹ in complete medium with mitomycin-C (Sigma-Aldrich)-treated E.G7-OVA at a 5:1 ratio in 6 well plates. Four days later, live cells were isolated on a lympholyte gradient (Cedarlane Laboratories Limited, Burlington, Ontario, Canada) and cultured in complete medium with 25 U ml⁻¹ IL-2 (R&D Systems, Minneapolis, Minn., USA) for two additional days. Cytotoxicity assays were performed using a CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, Wis., USA). 1×10⁴ E.G7-OVA cells per well were plated as target cells. Expanded splenocyte effector cells were incubated with the target cells at effector to target ratios of 1:1, 3:1, 10:1, and 30:1. Cultures were incubated in phenol-red free RPMI (Invitrogen) with 5% FBS (Omega) for 6 h at which point the cell culture supernatants were harvested. The lactate dehydrogenase (LDH) released from lysed cells was proportional to the resulting red formazan product, and was quantified using a microplate reader at 490 nm absorbance. The percentage of cytotoxicity was calculated according to the following equation: % Cytotoxicity=[(E−St−Se)/(M−St)]×100. Abbreviations are as follow; E=LDH release by effector-target coculture, St=spontaneous release by target cells, Se=spontaneous release by effectors, and M=maximal release by target cells.

Statistical Analysis

Data represented are mean±SEM. Data were analyzed for statistical significance using unpaired Student's t-test, 2-way ANOVA, or linear regression. Statistical analyses were done using GraphPad software version 5.01 for Windows (GraphPad Software, San Diego, Calif., USA). A p value <0.05 was considered statistically significant for these analyses.

Results Hp91 Induces Cytokine Release In Vivo

We have previously shown that exposure of DCs to an immunostimulatory peptide (ISP) named Hp91 in vitro leads to secretion of inflammatory as well as Th1 skewing cytokines[25]. To examine the adjuvant properties of Hp91 in vivo, serum cytokine responses were measured after intravenous (i.v.) injection of Hp91 into mice. Increased secretion of the Th1 cytokines IFN-γ, IL-12 (p70), as well as TNF-α was observed within 2 h of injection, with levels generally rising further over 24 h (FIG. 1).

Hp91 Enhances CD8 T Cell Responses to Peptide Antigen

Since the ISP Hp91 activates DCs and induces antigen-specific T cell responses in vitro[25], we tested whether Hp91 acts as an adjuvant to induce antigen-specific immune responses in vivo. The ISP Hp91 at both doses tested (250 μg and 500 μg) when co-administered with OVA peptides, caused a significant increase in the number of antigen-specific IFN-γ secreting T cells when splenocytes were restimulated with the CD8 epitope SIINFEKL (FIG. 2A), but not when restimulated with the CD4 epitope ISQAVHAAHAEINEAGR (FIG. 2B). Incomplete Freund's Adjuvant (IFA) a known stimulator of cell-mediated immune responses, elicited strong cellular immune responses when splenocytes were restimulated with either the CD8 or CD4 epitope (FIG. 2A,B).

The OVA-I peptide (SIINFEKL) is recognized in the context of H-2K^(b) MHC-Class I molecules and is specific for CD8+ T cells. To further confirm that the observed immune response is an OVA-specific CD8+ T cell response, CD4+ or CD8+ cells were depleted from the splenocytes prior to setup up the ELISPOT assay. While greater than 95% of CD4+ cells were depleted, CD8+ depletion was not as complete; 20% of CD8+ cells remained in the cultures as observed by flow cytometry. As expected, the number of IFN-γ secreting cells was reduced to near background levels (OVA peptides/PBS) in the CD8+ depleted splenocyte populations (FIG. 2C). In contrast to the CD8+ depletion, the CD4+ depleted splenocytes from the Hp91-OVA immunized groups retained the ability to secrete IFN-γ in response to OVA-I peptide stimulation further supporting the involvement of CD8+ T cells following co-immunization with the ISP Hp91 and OVA peptide showing that the ISP Hp91 causes activation and proliferation of antigen-specific CD8+ T lymphocytes in vivo.

To test whether lower doses of Hp91 would suffice as adjuvant for immunization, titrated doses of Hp91 were co-injected with OVA peptide to determine the minimum injection dose required for a significant increase in antigen-specific IFN-γ secreting T cells. As expected, a dose response was observed (FIG. 2D). However, with only 5 mice per group, a significant increase in IFN-γ secreting T cells was observed by ELISPOT only in the group receiving an Hp91 dose of 250 mg, suggesting 250 μg is an optimal dose when immunizing small groups of mice. Subsequent experiments showing 500 μg of Hp91 were conducted prior to the titration experiment.

The ISP Adjuvant Effect is Sequence Specific

To test if the in vivo adjuvant effect is related to the sequence of Hp91 or if any peptide will cause similar effects, we used a control peptide named Hp121. Hp121 is also derived from HMGB1 B-box and has the same length, a similar charge, and isoelectric point as Hp91. Hp121 does not cause activation of human DCs [25] or mouse BM-DCs in vitro. Mice were immunized s.c. with OVA-I peptide co-injected with either Hp91, Hp121 or PBS control. Mice immunized with Hp91/OVA-I peptide showed a significantly increased number of INF-γ secreting cells as compared to the Hp121/OVA-I peptide and PBS/OVA-I peptide immunized mice using freshly isolated as well as expanded splenocytes (FIG. 2E). No significant increase was observed between the Hp121/OVA peptide and PBS/OVA peptide immunized groups.

Hp91 Induces Th1-Type Immune Response In Vivo

Since IL-2 is critical for the activation, survival, and proliferation of T lymphocytes, we tested whether IL-2 secretion is increased in Hp91/OVA peptide immunized mice. Freshly isolated splenocytes were cultured overnight in presence of OVA peptide and culture supernatants were analyzed for IL-2 (FIG. 3) and IL-4, which may indicate the induction of a Th2 type immune response by ELISA. The highest IL-2 secretion was observed in OVA-I restimulated splenocytes from mice immunized with Hp91/OVA peptides (FIG. 3A), which showed higher IL-2 secretion compared to mice vaccinated with whole OVA protein/Hp91 or OVA peptide/IFA, both of which were also significantly increased as compared to the PBS/OVA control. After exposure to the MHC-Class II specific OVA-II peptide, splenocytes from Hp91/OVA peptide immunized mice showed a low but significant increase in IL-2 secretion as compared to PBS/OVA immunized mice (FIG. 3B). IL-4 was not detected in the splenocytes cultures at any of the conditions, though it was detected in the ConA stimulated positive control.

Hp91 Elicits Antibody Responses to Soluble Protein

Immunization using OVA protein in context of Hp91 promoted an antibody response to OVA which was dominated by the IgG1 isotype (FIG. 4A, 4B), Two-fold serial dilutions of the Hp91/OVA protein group show similar serum titers for the group (FIG. 4C). Minimal increase in IgG2b and IgM OVA specific antibodies was detected. Immunization using OVA peptides (OVA-I and OVA-II) together with Hp91 did not induce antibody responses (FIG. 4A).

Co-Administration of Hp91 with Antigen Induces CTL Responses

To further investigate whether OVA-specific cytotoxic T lymphocytes (CTL) responses were induced by immunization with the ISP Hp91, splenocytes from immunized mice were assessed for their ability to lyse OVA-expressing E.G7-OVA cells. The strongest killing was observed using effector splenocytes from mice immunized with Hp91/OVA peptides (FIG. 5), which was even higher than of splenocytes from IFA/OVA peptide immunized mice. When data were analyzed by linear regression and slopes were compared, the percent killing of target cells by effectors cells from mice immunized with Hp91/OVA peptides was significantly higher than the PBS control group;

Discussion

Although subunit vaccines promise to be less toxic, many are poorly immunogenic when administered without adjuvant. Alum, though FDA-approved, generates a weak Th1 response with a questionable safety profile. Thus, there is a great need for safer and more potent adjuvants [1-3].

We have previously shown that the 18 amino acid long ISP Hp91, is a stimulus for human DCs with the ability to generate a Th1-type immune response in vitro [25]. Here we demonstrate that Hp91 acts as adjuvant in vivo; inducing cellular immune responses to peptide and both cellular and humoral immune responses to protein antigen. The CD8 immune response was strong, since no in vitro expansion of splenocytes was needed to obtain a significant response as is commonly performed when testing vaccine responses. We show that the ISP Hp91 acts as an immune adjuvant to induce antigen-specific CD8 T cell responses in vivo. In addition, the immunostimulatory effects of Hp91 are related to its sequence, as the HMGB1 control peptide, Hp121, while matching Hp91 in length, isoelectric point, and charge, failed to induce cellular immune response.

The cytokine profile induced by an immune adjuvant plays an important role in the polarization of the immune response. The data show that co-immunization with the ISP Hp91 and OVA peptides as well as OVA protein results in OVA-specific secretion of IL-2, suggesting that immunized mice are able to mount an adaptive immune response that activates T cells to synthesize and secrete IL-2 for in vivo proliferation of OVA-specific effector T cells. Interestingly, IL-4 levels were undetectable. Intravenous administration of Hp91 resulted in increased secretion of the Th1 cytokines IFN-γ and IL-12 (p70) associated with cell-mediated immunity. This together with the measured IFN-γ secretion by the T cells along with undetectable IL-4 suggests that Hp91 induces a Th1-type of immune response in vivo. We also show that immunization with Hp91/OVA peptide elicited stronger CTL responses than IFA/OVA.

We show that immunization using OVA protein mixed with Hp91 also induced humoral immune responses. This is very promising for future development of this novel adjuvant, as it could be used for prophylactic vaccination against infectious disease.

The ability of Hp91 to induce antigen-specific cell mediated, Th1 immune response may make Hp91 suitable as an adjuvant in cancer immunotherapies as well as vaccines against infectious diseases caused by intracellular bacteria [4] or viruses [42,43]. Some of the adjuvants that are being evaluated in clinical and preclinical settings like CD40L [44] and poly(I:C) synthetic double stranded RNA [45], act on myeloid DCs and have shown promising results for tumour immunotherapy [46], emphasizing the importance of activating myeloid DCs. As a peptide adjuvant that acts directly on myeloid DCs [25], the peptides of the invention have several advantages. They can be made synthetically, are inexpensive, can be produced in high quantities at GMP quality, and they can also be genetically engineered to DC targeting molecules like DEC-205 which promotes strong immune responses when linked to a DC stimulatory molecule [47,48]. Since the tested doses of Hp91 have shown no adverse effects in mice to date, this current data suggest peptide should be well-tolerated for use in vaccines.

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Example 2 Materials and Methods Animals

C57BL/6 mice were purchased from Charles River Laboratories (Boston, Mass., USA). TLR4−/− and IL1R−/− mice were purchased from The Jackson Laboratories (Bar Harbor, Me., USA). IFNαβR−/− mice were purchased from B&K Universal (England, UK). MyD88−/− and TLR7−/− mice were a gift from S. Akira (Osaka University, Osaka, Japan) and backcrossed for 10 generations onto the C57BL/6 background. Mice were bred and maintained at the Moores UCSD Cancer Center animal facility and all animal studies were approved by the Institutional Animal Care and Use Committee of UCSD and were performed in accordance with the institutional guidelines.

Reagents

Phenylarsine oxide and chlorpromazine (clathrin-mediated endocytosis inhibitors), sodium azide (energy-dependent endocytosis inhibitor), nystatin (caveolin-mediated endocytosis inhibitor), latrunculin B (phagocytosis inhibitor), amiloride (micropinocytosis inhibitor), and Dynasore (dynamin inhibitor) were purchased from Sigma-Aldrich (St. Louis, Mo.) as endocytosis inhibitors. The p38 MAPK-specific inhibitor, SB203580, and the NFκB inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), were purchased from Sigma-Aldrich. The MEK1 inhibitor, PD98059, was purchased from Cell. Signaling Technology (Danvers, Mass.). As many of these inhibitors required solubilization in DMSO, DMSO was used as a negative control.

Peptides

The peptides, including Hp91 (DPNAPKRPPSAFFLFCSE), Hp121 (SIGDVAKKLGEMWNNTAA), scrambled Hp91 (ASLAPPFPNCFDPKSREF), and SIINFEKL were all purchased from GenScript Corp (Piscataway, N.J., USA) and CPC Scientific (San Jose, Calif., USA). Peptides were synthesized with an N-terminal biotin or fluorescent tag (Cp488). Peptides were routinely synthesized with greater than 95% purity. Peptides were dissolved in RPMI or PBS for in vitro and in vivo experiments respectively.

Cell Lines

The J774 cell line was a gift from Maurizio Zanetti (UCSD) and was cultured in RPMI 1640 medium (Invitrogen, Carlsbad, Calif.), supplemented with 10 mmol HEPES (GIBCO-BRL Life Technologies, Grand Island, N.Y.), penicillin (100 U/mL)-streptomycin (100 μg/mL)-L-glutamine (2 mM) (GIBCO-BRL), and 10% (vol/vol) fetal calf serum (Omega Scientific, Tarzana, Calif.). The RAW 264.7 cell line was a gift from Dong-Er Zang (UCSD) and was cultured as above, except with 5% (vol/vol) fetal calf serum (Omega).

Generation of Human Monocyte-Derived DCs

Peripheral blood mononuclear cells were isolated from the blood of normal volunteers over a Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient. Anonymous blood was purchased from the San Diego Blood Bank; therefore, no institutional review board approvals were necessary. To generate DCs, peripheral blood mononuclear cells were allowed to adhere to culture plates for 1 hour. The nonadherent cells were washed off, and the adherent cells were cultured in RPMI 1640 medium (Invitrigen) supplemented with 50 mmol 2-mercaptoethanol (Sigma), 10 mmol HEPES (GIBCO-BRL), penicillin (100 U mL⁻¹)-streptomycin (100 μg mL⁻¹)-L-glutamine (2 mM) (GIBCO-BRL), and 1% (vol/vol) human plasma (Valley Biomedical, Winchester, Va.) supplemented with GM-CSF (1000 U mL⁻¹) (Bayer HealthCare Pharmaceuticals, Wayne, N.J.), and interleukin-4 (100 U mL⁻¹) (IL-4; R and D Systems, Minneapolis, Minn.) at days 0, 2, and 4. Immature human DCs (iDCs) were harvested on days 5-7.

Generation of Mouse Bone Marrow-Derived DCs

Bone marrow-derived DCs (BM-DCs) were prepared from C57BL/6 and knockout mice, as described by Inaba et al (36) with minor modifications. Briefly, single bone marrow cell suspensions were obtained from femurs and tibias and depleted of lymphocytes, granulocytes, and Ia+ cells by incubating with a mixture of monoclonal antibodies (mAbs; anti-CD4, anti-CD8, anti-B220/CD45R, and anti-Ia) (antibody hybridomas were a gift from Ralph Steinman (Rockefeller)) and low-toxicity rabbit complement (Pel Freez Biologicals, Rogers, Ark.) for 60 minutes at 37° C. Cells were resuspended at a concentration of 10⁶ cells mL⁻¹ in RPMI 1640 medium (Invitrogen) supplemented with 10 mmol HEPES (GIBCO-BRL), penicillin (100 U/mL)-streptomycin (100 μg/mL)-L-glutamine (2 mM) (GIBCO-BRL), 5% (vol/vol) fetal calf serum (Omega), and 10 ng nor recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (J558L GMCSF-secreting cells were a gift from Ralph Steinman). Fresh complete medium containing GM-CSF was added on days 2 and 4 of culture. Cells were collected for the experiments on days 5-7.

Confocal Microscopy

Immature human DCs (10⁵) were pre-cooled on ice and subsequently incubated for 30 minutes on ice in culture medium with biotinylated Hp91 or Hp121 to allow peptide binding. Cells were washed and then incubated for the indicated time at 37° C. Cells were cytospun (Shandon Cytospin 2 centrifuge) onto glass slides, fixed, permeabilized with acetone, and stained with Streptavidin-Alexa 488 (Invitrogen) to visualize biotinylated peptides and Hoechst 33258 DNA stain (Invitrogen). Cells were imaged on a Zeiss LSM confocal microscope.

Flow Cytometry

For most experiments, iDCs were pre-cooled on ice for 30 minutes. Cells were subsequently incubated for the indicated times and temperatures in culture medium with biotinylated peptides. Cells were washed, permeabilized with Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, N.J.) stained with Streptavidin-Alexa 488 (Invitrogen), and analyzed by flow cytometry. For experiments with endocytosis inhibitors, cells were pre-treated for 30 minutes with the indicated inhibitors prior to incubation with the biotinylated peptides. For experiments with J774 macrophages, cells were pre-cooled on ice, pre-treated with 30 minutes with the indicated inhibitors, and subsequently incubated for 30 minutes with fluorescently-labeled Hp91 (Cp488-Hp91). Cells were immediately analyzed by flow cytometry.

Stimulation of iDCs and BM-DCs

At days 5-7 of culture, iDCs were either left untreated or were stimulated with indicated doses of peptide. For inhibition experiments, immature human DCs were pre-treated with the indicated doses of SB20358, PD98059, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), or DMSO control for 30 minutes prior to stimulation. For experiments with human DCs, supernatants were analyzed by ELISA (eBioscience, Inc. San Diego, Calif.), according to the manufacturer's instructions, 48 h after stimulation. For experiments with mouse BM-DCs, supernatants were analyzed by ELISA (eBioscience), 24 h after stimulation.

Immunizations and Splenocyte Preparation

Mice were immunized s.c. with 50 μg of SIINFEKL peptide. The SIINFEKL peptide was co-administered with either PBS, Hp91 (250 μg), or scrambled Hp91 (250 μg). Peptides were dissolved in PBS for all immunizations. Mice were boosted two weeks later and spleens and blood were collected one week after the final immunization. Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70 mm nylon cell strainer (BD Biosciences). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, Ind.) and the splenocytes were subsequently resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10 mmol HEPES (GIBCO-BRL), penicillin (100 U/mL)-streptomycin (100 μg/mL)-L-glutamine (2 mM) (GIBCO-BRL), and 5% (vol/vol) fetal calf serum (Omega).

Enzyme-Linked Immunospot Assay

Freshly isolated splenocytes were plated in duplicate to wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (Millipore, Millerica, Mass., USA) that had been previously coated overnight with 5 μg ml⁻¹ monoclonal anti-mouse IFN-γ antibody (Mabtech, Stockholm, Sweden). Splenocytes were cultured overnight at 37° C. with 2.5 μg ml⁻¹ SIINFEKL peptide, 5 μg ml⁻¹ concanavalin A positive control (Sigma), or left unstimulated (medium only). After 18 h, culture supernatants were collected for cytokine analysis and ELISPOT plates were developed using 1 μg ml⁻¹ biotinylated anti-mouse IFN-γ antibody (Mabtech), Streptavidin-HRP (Mabtech), and TMB Substrate (Mabtech). The plate was scanned and the spots were counted using an automated ELISpot Reader System (CTL ImmunoSpot, Shaker Heights, Ohio, USA).

Cytokine Release Assay

To measure cytokine release from immunized mice, splenocytes were cultured overnight with 2.5 μg ml⁻¹ SIINFEKL peptide, 5 μg ml⁻¹ concanavalin A positive control (Sigma), or left unstimulated (medium only). After 18 h, cell culture supernatants were collected and analyzed for the presence of IL-2 by ELISA (eBioscience).

Immunoblotting

Mouse J774 or RAW 264.7 macrophages stimulated with Hp91 or LPS for 20, 40, or 60 minutes were lysed for 20 minutes on ice in RIPA lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 5 mM EDTA supplemented with 1 mM phenylmethylsulfonyl fluoride, Halt phosphatase inhibitor (Thermo Fisher Scientific), 1 mM sodium vanadate, 1 mM sodium fluoride, and complete protease inhibitor cocktail (Roche). Protein concentration was determined with the DC (Detergent Compatible) protein assay (Bio-Rad). The lysates were snap-frozen and stored at −80° C. Equal amounts of protein lysates were separated by gel electrophoresis with the use of a NuPAGE Novex 4%-12% Bis-Tris Midi Gel (Invitrogen) and transferred to polyvinylidene fluoride membranes (Bio-Rad). Membranes were washed with 1×TBST (Tris-Buffered Saline Tween-20) and blocked for 1 hour at room temperature in 5% milk/TBST. Membranes were probed overnight for phospho-p38, phospho (p)-interferon regulatory factor 3 (IRF3), p38, IRF3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or β-actin (Cell Signaling Technology). The next day, membranes were washed with 1×TBST and incubated with goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.) diluted in 5% milk/TBST for 1 hour at room temperature. Antibodies were detected with the use of either an enhanced chemiluminescence detection kit (GE Healthcare, Piscataway, N.J.) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, Ill.). In some experiments, cells were pre-treated with the endocytosis inhibitor Dynasore.

Statistical Analysis

Data represented are mean±SEM. Data were analyzed for statistical significance using unpaired or paired Student's t-tests. Statistical analysis was done using GraphPad software version 5.01 for Windows (GraphPad Software, San Diego, Calif., USA). A p value <0.05 was considered statistically significant.

Results Hp91 Enters Dendritic Cells Via Clathrin-Mediated Endocytosis

To gain insight into the mechanism of action of the DC stimulatory peptide Hp91, we investigated its interaction with DCs. DCs were exposed to Hp91 or a control peptide, Hp121, which is also derived from HMGB-1 and has the same length, a similar charge and isoelectric point as Hp91. Confocal microscopy showed that Hp91 was detected within DCs within 5 minutes, while the control peptide Hp121 was not detected, even after 30 minutes at 37° C. (FIG. 17A). We observed a dose dependent uptake of Hp91 by flow cytometry (FIG. 17B), which appears to plateau between 10 and 30 minutes (FIG. 17C). Uptake of Hp91 is sequence specific, as the control peptide Hp121 was not taken up by DCs (FIG. 17D, 17E). Although a scrambled version of Hp91 was taken up by DCs, it was at a much lesser extent than Hp91 (FIG. 17D, 17E), and mice immunized with the scrambled Hp91 sequence failed to mount an antigen-specific immune response as compared to Hp91 (FIG. 17F, 17G).

Cell penetrating peptides are preferentially taken up via endocytosis, at 37° C., or transcytosis (37), which occurs at 16° C. We tested the temperature at which Hp91 was taken up by DCs to determine the method of uptake. DC uptake of Hp91 occurred at 37° C., but not 16° C., or 4° C. (FIG. 18A, 18B), suggesting that uptake occurs via an energy dependent process, such as endocytosis. Endocytosis pathways include phagocytosis, macropinocytosis, clathrin-mediated, or lipid-raft/caveolin-mediated. The mechanism of uptake was determined using specific inhibitors of these endocytosis pathways. Latrunculin B (LatB), a specific phagocytosis inhibitor, while completely abrogating uptake of Dextran FITC, did not significantly reduce uptake of Hp91 (FIG. 18C). Phenylarsine oxide and chlorpromazine, both clathrin-mediated endocytosis inhibitors, significantly reduced the uptake of Hp91, suggesting that uptake occurs via clathrin-mediated endocytosis (FIG. 18D). Sodium azide, which generally tests for energy-dependent endocytosis, inhibited Hp91 uptake by approximately 40% (FIG. 18D), though not significantly. When using a fluorescently labeled version of Hp91 (Cp488), sodium azide significantly inhibited Hp91. uptake. In contrast, the lipid-raft/caveolin-mediated endocytosis inhibitor, nystatin, and the macropinocytosis inhibitor, amiloride, failed to significantly reduce uptake of Hp91 (FIG. 18D), suggesting uptake is not via lipid rafts, nor macropinocytosis.

Hp91 Induces IL-6 Secretion in DCs Via p38 MAPK and NFκB

We have previously shown that the p38 MAPK inhibitor, SB203580, down-regulated HMGB1 B box-induced secretion of IL-6 (38). Pretreatment of human DCs with SB203580, a p38 MAPK inhibitor, and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), an NF-κB inhibitor, significantly reduced the Hp91-induced IL-6 secretion (FIG. 19). In contrast, PD98059, a MEK1 inhibitor, failed to significantly reduce IL-6 secretion (FIG. 19). These results suggest that Hp91 causes DC activation in a p38 and NF-κB dependent manner.

Hp91-Mediated Activation of DCs is Dependent on MyD88, TLR4, and IFNαβR

As clathrin-mediated endocytosis is receptor-mediated, we set out to identify the receptor(s) for Hp91. Several receptors have been implicated in mediating the responses to the parent molecule HMGB1, including TLR4 (19-22, 24, 26). To identify the receptor(s) involved in activation of DCs, mouse BM-DCs were generated from wildtype and knockout mice and cells were exposed to Hp91. IL-6 secretion was strongly and significantly reduced in BM-DCs from MyD88 and TLR4 knockout mice (KO) (FIG. 20A), whereas IL-6 production from BM-DCs generated from TLR7 KO mice was comparable to wild type (FIG. 20A). In addition, IL-6 secretion in IFNαβR knockout mice was significantly reduced (FIG. 20A). No change was observed in IL-6 secretion from IL-1R knockout mice.

Involvement of TLR4 and MyD88 and their downstream pathways was further confirmed by immunoblotting for phospho-p38. Phospho-p38 (p-p38) was upregulated after 40-60 minutes of Hp91 stimulation (FIG. 20B) in 4 independent experiments. In some, but not all experiments, upregulation of p-p38 was observed as early as 20 minutes after Hp91 stimulation.

Hp91-Mediated Activation of DCs is Clathrin- and Dynamin-Dependent

In LPS signaling of macrophages, TLR4 is endocytosed in a dynamin-dependent manner for downstream signaling (39), so we tested if pre-treatment of macrophages with clathrin- and dynamin-mediated endocytosis inhibitors would reduce Hp91-mediated IL-6 secretion. Similar to with LPS, pre-treatment with the dynamin-dependent endocytosis inhibitor, Dynasore, significantly reduced Hp9′-induced IL-6 secretion (FIG. 20C). In addition, a significant reduction in IL-6 was observed after blocking clathrin-mediated endocytosis with phenylarsine oxide (FIG. 20C). Interestingly, no reduction in IL-6 was observed after pre-treatment with chlorpromazine. As we had observed a reduction in Hp91-mediated IL-6 secretion in human DCs following pretreatment with the p38 and NF-κB inhibitors, SB203580 and TPCK, in human DCs, we tested if a similar reduction in IL-6 would be observed in mouse cells. As was seen in human cells, SB203580 and TPCK significantly lowered than the amount of Hp91-stimulated IL-6 secreted from mouse macrophages (FIG. 20C), further confirming the involvement of p38 and NF-κB in Hp91 signaling. Furthermore, we demonstrated that the dynamin-dependent endocytosis inhibitor, Dynasore, significantly inhibited uptake of Hp91 (FIG. 20D).

As a decrease in Hp91-induced IL-6 secretion was observed in IFNαβR knockout BM-DCs (FIG. 20A), we set out to investigate if the MyD88-independent, TRAM/TRIF signaling pathway was activated by probing for phosphorylation of the downstream interferon regulatory factor 3 (IRF3) transcription factor. Phospho-IRF3 (pIRF3) was upregulated after Hp91 stimulation (FIG. 20E). In addition, blocking endocytosis with Dynasore reduced the phosphorylation of IRF3, suggesting that Hp91 uptake is required for MyD88-independent interferon regulatory factor 3 signaling.

A Proposed Mechanism of Hp91 Signaling

We used the BM-DC knockout IL-6 data, immunoblots, and small molecular inhibitor data to develop a proposed mechanism of Hp91 signaling (FIG. 21)

Discussion

As shown by confocal microscopy, Hp91 binds cells within 30 minutes on ice, as visible by a rim around the cells, and is taken up into the cells very rapidly in a sequence specific manner, as Hp121 does not enter cells. Scrambling the amino acids of Hp91 results in a great reduction of binding/uptake by cells, indicating that it is neither the total charge or total hydrophobicity that is important. Rather, we hypothesized that a particular sequence of amino acids is critical for the binding and uptake of peptide into cells, and that the region of the peptide responsible for activity could be investigated by testing shorter, overlapping peptides for their dendritic cell uptake as well as in vitro and in vivo immunostimulatory activity. In Example 3, we identify such a short immunostimulatory peptide, UC1018, that activates DCs and acts as adjuvant in vivo.

Using a panel of endocytosis inhibitors, we demonstrate that Hp91 is taken up in a clathrin-dependent manner. As clathrin-mediated endocytosis is receptor mediated, we explored possible receptors for Hp91.

TLR2 and TLR4, in particular, are interesting receptors in that they appear to bind many ligands with a large amount or promiscuity, and it has been difficult to find many structural similarities among the ligands. Matzinger theorizes that it is hydrophobic portions (hyppos) of molecules that interact with receptors such as TLR2 and TLR4 (42).

We show using knockout mice that Hp91-stimulated activation of DCs is dependent on TLR4 and MyD88. We further show, via phosphorylation of p38 and using p38 inhibitors, that Hp91 activates the MyD88-dependent signaling pathway. We show via Hp91-induced phosphorylation of IRF3 that Hp91 does signal through the interferon pathway. Furthermore, we demonstrate that Hp91 uptake is required for phosphorylation of this MyD88-independent pathway.

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Example 3 Materials and Methods Animals

Female C57BL/6 mice 8-12 weeks of age were used for experiments. All mice were purchased from Charles River Laboratories (Boston, Mass., USA) and housed at the Moores UCSD Cancer Center animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee of UCSD and were performed in accordance with the institutional guidelines.

Peptides

The peptides, including Hp91 (DPNAPKRPPSAFFLFCSE), UC18 (DPNAPKRP), UC411 (APKRPPSA), UC714 (RPPSAFFL), UC718 (RPPSAFFLFCSE), UC1018 (SAFFLFCSE), and SIINFEKL were all purchased from GenScript Corp (Piscataway, N.J., USA) and CPC Scientific (San Jose, Calif., USA). UC1018 mutated peptides (shown in FIG. 26) were synthesized at CPC Scientific. For uptake experiments and immunizations, peptides were synthesized with an N-terminal biotin. For DC stimulation experiments, peptides were synthesized with an N-terminal acetyl group. Peptides were routinely synthesized with greater than 95% purity. Unless otherwise stated, all peptides were dissolved in RPMI.

Cells

The murine melanoma B16.F1 cell line, transduced with a cDNA encoding the chicken ovalbumin gene (B16cOVA), was a gift from Richard Vile (Mayo) and were previously described (15). The cells were cultured in Dulbecco's modification of Eagle's Medium (Mediatech, Manassas, Va.), supplemented with 2 mM L-glutamine (GIBCO-BRL Life Technologies, Grand Island, N.Y.), 10 mmol HEPES (GIBCO-BRL), penicillin (100 U/mL)-streptomycin (100 μg/mL) (GIBCO-BRL), and 10% (vol/vol) fetal calf serum (Omega). To induce apoptosis of B16 cells, the cells were treated with 0.5 mg ml⁻¹ mitomycin C in DMEM media for 60 minutes at 37° C. Cells were washed twice in warm DMEM and put back to culture overnight in DMEM supplemented as above.

Maleimide Conjugation Reactions

Hp91 peptide monomers, capped at the thiol group of the cysteine, were generated using an N-Ethylmaleimide (NEM) (Thermo Scientific) conjugation reaction. Briefly, Hp91 was dissolved in PBS and reacted for 2 h at RT in the presence of NEM. Hp91 peptide maleimide dimers, cross-linked at the thiol group of the cysteine, were generated using a Bis-maleimidoethyleneglycol (BM(PEG)₂) (Thermo Scientific) conjugation reaction. Briefly, Hp91 was dissolved, in PBS/EDTA and reacted for 1 h at RT in the presence of BM(PEG)₂. Excess NEM or BM(PEG)₂, respectively, was removed by dialysis (2K MWCO cassette, Thermo Scientific). Un-reacted, mock peptide controls were generated under identical reaction and dialysis conditions, while excluding the NEM or BM(PEG)₂ reagent. Reagents, glassware, and reaction products were endotoxin-free as determined by the manufacturer or a limulus amoebocyte assay (LAL) (Cambrex Corporation, East Rutherford, N.J.) tested according to manufacturer's instructions. Peptides with a biotin at the N-terminal group were used for binding/uptake experiments. Peptides with an acetyl at the N-terminal group were used for DC stimulation experiments.

Spontaneous Oxidation and Dithiothreitol-Reduction of Hp91

Hp91 was incubated in PBS at RT for 24 to 96 h to allow spontaneous oxidation of cysteine residues. In some experiments, the peptide was incubated for 30 min+/−10 mM dithiothreitol (DTT) (Thermo Scientific) and/or 1% H₂O₂ (Thermo Fischer Scientific) to reduce or further oxidize the peptide, respectively.

Generation of Human Monocyte-Derived DCs

Peripheral blood mononuclear cells were isolated from the blood of normal volunteers over a Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient. Anonymous blood was purchased from the San Diego Blood Bank; therefore, no institutional review board approvals were necessary. To generate DCs, peripheral blood mononuclear cells were allowed to adhere to culture plates for 1 hour. The nonadherent cells were washed off, and the adherent cells were cultured in RPMI 1640 medium (Invitrigen) supplemented with 50 mmol 2-mercaptoethanol (Sigma), 10 mmol HEPES (GIBCO-BRL), penicillin (100 U mL⁻¹)-streptomycin (100 μg mL⁻¹)-L-glutamine (2 mM) (GIBCO-BRL), and 1% (vol/vol) human plasma (Valley Biomedical, Winchester, Va.) supplemented with GM-CSF (1000 U mL⁻¹) (Bayer HealthCare Pharmaceuticals, Wayne, N.J.), and interleukin-4 (100 U mL⁻¹) (IL-4; R and D Systems, Minneapolis, Minn.) at days 0, 2, and 4. Immature human DCs (iDCs) were harvested on days 5-7.

Stimulation of DCs

At days 5-7 of culture, immature dendritic cells were either left untreated or were stimulated with Hp91 maleimide reaction products, Hp91 controls, or short peptides as indicated and incubated at 37° C. Supernatants were collected 48 h after stimulation and were analyzed for IL-6 by ELISA (eBioscience), according to the manufacturer's instructions.

Peptide Binding/Uptake Studies

Immature DCs were incubated for the indicated times and temperatures in culture medium with biotinylated peptides, biotinylated peptide reaction products or unreacted, mock controls. Cells were washed, permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry.

Peptide Immunization and Spleen Cell Preparation

Mice were immunized s.c. with 50 μg of SIINFEKL peptide. The SIINFEKL peptide was co-administered with either PBS, Hp91 (250 μg), UC714 (129 μg), or UC1018 (142 μg). Hp91, UC714, and UC1018 peptide doses were equimolar. Mice were boosted two weeks later and spleens and blood were collected one week after the final immunization. Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70 mm nylon cell strainer (BD Biosciences, Franklin Lakes, N.J., USA). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, Ind., USA) and the splenocytes were subsequently resuspended in complete medium (RPMI 1640 with 10% FBS, L-glutamine, penicillin, streptomycin, and HEPES). Peptides were dissolved in PBS for all immunizations.

Enzyme-Linked Immunospot Assay

Freshly isolated splenocytes were plated in duplicate to wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (Millipore, Millerica, Mass., USA) that had been previously coated overnight with 5 μg ml⁻¹ monoclonal anti-mouse IFN-γ antibody (Mabtech, Stockholm, Sweden). Splenocytes were cultured overnight at 37° C. with 2.5 μg ml⁻¹ SIINFEKL peptide, 5 μg ml⁻¹ concanavalin A positive control (Sigma), or left unstimulated (medium only). After 18 h, ELISPOT plates were developed using 1 μg ml⁻¹ biotinylated anti-mouse IFN-γ antibody (Mabtech), Streptavidin-HRP (Mabtech), and TMB Substrate (Mabtech). The plate was scanned and the spots were counted using an automated ELISpot Reader System (CTL ImmunoSpot, Shaker Heights, Ohio, USA).

Cytokine Release Assay

To measure cytokine release from immunized mice, splenocytes were cultured overnight with 2.5 μg ml⁻¹ SIINFEKL peptide, 5 μg ml⁻¹ concanavalin A positive control (Sigma), or left unstimulated (media only). After 18 h, cell culture supernatants were collected and analyzed for the presence of IL-2 by ELISA (eBioscience).

Prophylactic B16 Tumor Challenge

Mice were immunized s.c. with 2×10⁵ apoptotic mitomycin-C (Sigma)-treated B16 cells co-administered with either PBS or UC1018 (142 μg). Mice were boosted twice, at 4 weeks and 6 weeks post-prime as above, and challenged s.c. into the flank with 2×10⁵ live B16 cells at one week post-boost. Mice were followed for tumor growth and survival. Tumor dimensions were measured using calipers and the tumor volume calculated using the following formula; volume=4/3π(a²×b). Mice were euthanized when tumor volume reached 1.5 cm³. Tumor survival curves were generated, wherein the day of euthanasia was considered as death.

HPLC

Peptides were evaluated by HPLC (Agilent 1100 Series, Software: Chemstation) at 211 nm (column: ZORBAX RP C18). Percent dimer was calculated as the area under the curve (AUC) of dimer/(AUC of monomer+AUC of dimer).

Circular Dichroism

CD spectra of peptides, dissolved in 75%/25% Trifluoroethanol (TFE, Sigma)/H₂O (vol/vol), were collected on an AVIV model 202 Circular Dichroism Spectrometer, under nitrogen, using a 1 mm pathlength quartz cuvette. These spectra were corrected by subtraction of a “solvent-only” spectrum and smoothed. The spectra are shown in mean residue ellipticity (θ_(mrw)). Peptides were analyzed at 200 μg ml⁻¹.

Statistical Analysis

Data represented are mean±SEM. Data were analyzed for statistical significance using unpaired or paired Student's t-test. Statistical analysis was performed using GraphPad software version 5.01 for Windows (GraphPad Software, San Diego, Calif., USA). A p value <0.05 was considered statistically significant.

Results

Hp91 Forms Spontaneous, Reversible Dimers

Cysteine residues, with an unprotected sulhydryl group, can form disulphide bridges. There is a cysteine at position 16 in Hp91 that could oxidize under certain conditions to form a peptide dimer with a second Hp91 molecule. Hp91 peptide, dissolved in PBS in the presence of oxygen, was incubated at RT for up to 96 h, and evaluated for the presence of dimers using HPLC. Hp91 dimers, running slower off the HPLC C18 column, were present at approximately 25% of the total peptide within 24 h. Dimers continued to form until measurements were stopped at 96 h, at which point greater than 80% of the peptide was in a dimer formation (FIG. 22A, 22B). To determine if this peptide dimer was stable or easily reversible, 24 h peptide dimers were allowed to form, and followed with a subsequent reduction in the presence of DTT. After 30 minutes at 10 mM DTT, all Hp91 peptide was found in a monomer state, as demonstrated by the reduction to a single HPLC peak (FIG. 22C).

As shown in Example 2, Hp91 binds and is endocytosed by DCs, as demonstrated by confocal microscopy and flow cytometry. Here we investigated if reducing or oxidizing the peptide would result in respective decreases or increases in uptake. There was no reason to believe that it would increase Hp91 activity. Compared to untreated peptide controls, uptake of DTT-reduced Hp91 peptide was reduced by approximately 50% (FIG. 22D). Addition of peroxide alongside the DTT maintained the peptide uptake at levels close to normal, where addition of peroxide to non-reduced peptide may enhance peptide uptake by a small amount. This data suggest that uptake of peptide may be enhanced by dimerization. This was a surprising, unexpected result.

Maleimide Dimers Enhance DC Activity

Since the DTT and peroxide appeared to have an affect on the uptake of Hp91, we generated capped peptide monomers and cross-linked peptide dimers using maleimide conjugation reactions and evaluated the conjugated peptides for DC uptake and activation. Mono- or bis-maleimides contain an imide group that reacts readily with the thiol group of cysteine to form a stable carbon-sulfur bond. We predicted that this stability would enable us to conduct more robust experiments than would be possible with DTT- and peroxide-treated peptides. NEM is a mono-maleimide that generates a capped peptide monomer. BM(Peg)₂ is a bis-maleimide that cross-links two peptides to form a dimer.

HPLC of the maleimide conjugation reaction products shows that the NEM capped monomer forms a single peptide peak, suggestive of close to 100% monomer, where the BM(PEG)₂ cross-linked dimer forms two peaks with an estimated 65% dimer (FIG. 23A). We evaluated the capped monomer for its ability to be taken up by DCs by incubating a biotinylated capped peptide, or control Hp91 peptide, with DCs and staining for uptake, as described above. Uptake was evaluated by flow cytometry and demonstrated that capping Hp91 and forcing it into a monomer state decreases its ability to be taken up by DCs by 27% (FIG. 23B). We tested whether the NEM capped peptide would similarly exert diminished immunostimulatory effects on DCs, we stimulated DC for 48 h with an acetylated capped Hp91 peptide, as compared to a control. As predicted, the Hp91 monomer exerts a reduced immunostimulatory effect on DC, as determined by a reduction in IL-6 secretion from DCs (FIG. 23C). Similar experiments concluded on the bis-maleimide dimers show that the dimer enhances uptake by 3-fold compared to controls (FIG. 23D), and enhances the immunostimulatory effects on DC, as shown by an approximately 10-fold increase in IL-6 secretion from stimulated DCs compared to controls (FIG. 23E). These results demonstrate that maleimide Hp91 dimers enhance uptake and cytokine secretion from DCs.

Activity of Hp91 Resides in C-Terminal Amino Acids

In an effort to identify the region of Hp91 required for DC binding and activation, overlapping 8-12 aa long peptides that span the length of Hp91 were synthesized and evaluated (FIG. 24A). DCs were incubated with the indicated short peptides and supernatants were tested after 48 hours for the presence of IL-6. While the C-terminal half of the peptide induced IL-6 at levels comparable to the full-length peptide, the N-terminal failed to induce cytokine secretion from DCs (FIG. 24B). Similar IL-6 secretion was observed from mouse BM-DCs stimulated with UC714 and UC718 peptides.

To test uptake of the short peptides, DCs were incubated with biotinylated versions of the short peptides for 30 minutes at 37° C. to allow uptake. Cells were fixed, permeabilized, stained with streptavidin-Alexa 488, and evaluated by flow cytometry. The 9 aa acid long peptide UC1018, which corresponds to the C-terminal half of Hp91, enhanced uptake by DCs over 3-fold compared to Hp91 controls (FIG. 24C, 24D). This was very surprising and unexpected. In contrast, UC18, UC411, and UC714, containing varying portions of the N-terminal 14 aa of Hp91, failed to be endocytosed by DCs (FIG. 24C, 24D).

The C-terminal half of Hp91 peptide appeared responsible for in vitro activity of Hp91, thus we set out to verify if the C-terminal domain was similarly responsible for in vivo activity. We have previously shown that full-length Hp91 peptide acts as adjuvant in vivo to induce antigen-specific immune responses. We hypothesized that either one or both of the C-terminal short peptides, UC714 and UC1018, would act as adjuvant to induce antigen-specific immune responses in vivo. One would expected that if the short peptides had adjuvant activity, it would be less than the 18mer. Mice were co-immunized with ovalbumin (SIINFEKL) peptide as antigen and equimolar amounts of UC714, UC1018, or Hp91. The CTD UC1018 induced a significant increase in the number of antigen-specific IFNγ-secreting T cells compared to PBS controls as measured by ELISPOT (FIG. 24E). These UC1018-induced immune responses were 4-fold stronger than our Hp91 positive control. This was extremely unexpected as it is half the length of the 18mer. UC714, though showing moderate increases in IFNγ-secreting T cells compared to PBS, showed only 50% of the IFNγ-secreting T cells observed with full-length Hp91 peptide (FIG. 24E). We further show that IL-2, which is critical for the activation, survival, and proliferation of T lymphocytes, was significantly enhanced in mice immunized with UC1018, at levels 4-fold higher even than Hp91 controls (FIG. 24F). This was unexpected.

In cancer treatment, an antigen-specific immune response becomes more meaningful if it translates into a survival advantage. As UC1018 peptide induced a significant antigen-specific immune response in vivo, we tested whether UC1018 could act as adjuvant in a prophylactic tumor challenge to induce anti-tumor immunity. Mice were co-immunized with apoptotic B16 melanoma cells and either PBS or UC1018 peptide. Mice were boosted twice and challenged one week post boost with live B16 melanoma cells. The melanoma model used in this study is highly lethal, such that within 7-10 days, the PBS control mice demonstrated tumor formation and rapid tumor growth, with the first mouse being sacrificed by day 1.6 post challenge due to a substantial tumor burden (FIG. 24G). In marked contrast to the PBS group, the UC1018 peptide immunized mice demonstrated a delay in tumor formation and slowing of tumor growth, significant as early as 11 days post challenge (FIG. 24G), indicating an induction of significant protective anti-tumor immunity in the UC1018 immunized mice. The mice were monitored for survival, and mice that received the UC 1018 prophylactic treatment demonstrated a striking and significant enhancement of survival, with 60% of mice tumor-free and alive at 75 days post tumor challenge (FIG. 24H). In contrast, all mice in the control group developed tumor with 100% of mice succumbing to tumor burden by 49 days post challenge (FIG. 24H).

C-Terminal Portion of Hp91 is Helical

As the CTD of Hp91, UC1018, is important for DC uptake and for exerting its immunostimulatory effects, we set out to evaluate the secondary structure of this peptide. Circular dichroism (CD) measures the difference in absorbance of right- and left-circularly polarized light. The phenomenon of CD is sensitive to the secondary structure of polypeptides and can be used between wavelengths of 190-250 nm to analyze a peptide for different structural types such as alpha helix, beta sheet, polyproline II helix, random coil, and more.

To perform the CD experiment, we dissolved peptides in a trifluoroethanol (TFE) buffer (75%/25% TFE/H₂0 by volume), which enhances polypeptide folding and structures by allowing hydrophobic portions to fold as they might in vivo. The CD spectrum of the CTD UC1018 showed negative ellipticity bands at 207 and 222 nm, with a positive ellipticity at 198 nm (FIG. 25). Such a spectra is characteristic of an alpha helix. The CD spectrum for the NTD UC18 showed a negative maximum at 202 mm (FIG. 25), which is close to the negative peak expected at 200 nm for a prolyproline II helix. A spectra with a negative peak around 200 DM is relatively ambiguous however, as random coils also display similar spectra. With some uncertainty, we conclude that the NTD UC 18 is a polyproline II helix, rather than random coil. This is based on the peptide sequence that contains two PXXP motifs, which are known to form polyproline H helices (118). The CD spectrum for the entire length of Hp91 remains more difficult to interpret. The negative peak near 205 for Hp91 (FIG. 25) may be an additive effect of the NTD and CTD spectra negative maximums.

Mutating Amino Acids can Strengthen Immunostimulatory Potential

UC1018 short peptide showed surprising immunostimulatory potential in vivo and acted as adjuvant, inducing anti-tumor responses. We hypothesized that we could mutate amino acids to kill activity and identify the amino acids critical for Hp91 activity. Surprisingly, replacing each of the C-terminal 4 amino acids of UC1018 with an alanine resulted in strengthened IL-6 secretion from DCs (FIG. 26). This was completely unexpected. Replacing the phenylalanine residues with serines did not have much affect on the immunostimulatory activity of Hp91.

Discussion

There is a great need for safer and more potent adjuvants (16, 17). We have previously shown that the 18 amino acid long immunostimulatory peptide (ISP) Hp91 is a potent stimulus for human DCs with the ability to generate a Th1-type immune response in vitro (7). We have recently shown that Hp91 acts as adjuvant in vivo; inducing cellular immune responses to peptide and both cellular and immoral immune responses to protein antigen (8). The results presented here characterize the structural basis for Hp91 activity, identifying a new ISP, UC1018, that acts as adjuvant in vivo and demonstrating that Hp91 dimerization enhances activity. In addition, we show that amino acid mutations of UC1018 maintain, if not strengthen immunostimulatory potential of the peptide.

We set out to investigate what structures and amino acid domains were responsible for the activity of the Hp91 immunostimulatory peptide and adjuvant. After determining that Hp91 formed spontaneous dimers in the presence of air, we tested whether these dimers would affect Hp91's activity. While we hypothesized that Hp91 dimerization at the cysteine might prevent proper binding and uptake into dendritic cells, surprisingly we discovered that dimers actually enhanced peptide uptake. We show in Example 2 that activity of Hp91 is dependent on TLR4. Toll-like receptors, such as TLR4, form homo- or hetero-dimers with other members of their protein family. Such homodimers of TLR4 are thought to be necessary for recruitment of adaptor proteins and subsequent signaling, as the Mal and TRAM adaptors are predicted to bind at the TLR4 dimer interface (18). It is possible that dimerization of Hp91 peptide promotes TLR4 dimerization, adaptor recruitment, and downstream signaling leading to activation of NFκB and IRF-3.

It is surprising to us that the dimers would enhance Hp91 activity. We could imagine the peptide binding to a groove in a single protein or receptor. It is hard to picture that a small peptide, even in dimer form, would be large enough to simultaneously bind both subunits of the TLR4 homodimer.

In Example 2, we showed that the scrambled version of Hp91 is not taken up by DCs, suggesting that it is neither the total charge nor hydrophobicity of the peptide that is important for Hp91 activity. Rather, it is a particular sequence of amino acids that leads to the immunostimulatory activity and binding of peptide to cells. In this example we determine the region of the peptide responsible for activity by testing shorter, overlapping peptides for their binding. Hp91 contains two PXXP motifs at the N-terminal half, present in short peptides UC18 and UC411, that we originally hypothesized may be responsible for the cell permeability and perhaps activity as cell permeable peptides often contain proline-rich regions (19, 20). To our surprise, it was the C-terminal end, not the N-terminal end of the peptide with activity, We demonstrate here that the C-terminal domain of Hp91 (the region that includes FCSE) induces cytokine release from DCs (UC718 peptide) and binds or is taken up by dendritic cells (UC1018). In addition, we show that the CTD, UC1018, is sufficient to act as adjuvant in vivo, inducing cellular, antigen-specific immune responses. Very surprisingly and unexpectedly, we demonstrate that UC1018, despite being only half the length of Hp91, is taken up by DCs to a much greater extent and is a 3-fold stronger adjuvant in vivo, as measured by IFN-γ ELISPOT and IL-2 cytokine release. Historically, shortening the HMGB1 protein to the B box domain reduced immunostimulatory activity, and similarly shortening the B box domain to the Hp91 peptide reduced the immunostimulatory activity further. Here, we surprisingly and unexpectedly show that shortening the sequence further, to UC1018, actually strengthens the immunostimulatory activity of the peptide.

We expected that mutating the amino acids of UC1018 would kill the immunostimulatory activity and help us identify the amino acids critical for activity. Surprisingly, we show here that mutating residues of the UC1018 peptide can result in increased immunostimulatory activity.

In summary, in this example we have investigated the activity of Hp91 immunostimulatory peptide, whose structure-function relationships had not been clear before now. We show that Hp91 dimerization enhances activity and that the helical C-terminus, UC1018, has DC binding and adjuvant activity that is stronger than full-length Hp91. We show that it is possible to potentiate the adjuvant effect by mutating the C-terminal amino acids of UC1018.

REFERENCES FOR EXAMPLE 3

-   1. Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B,     Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune     adjuvant released by necrotic cells, EMBO Rep. 2004; 5(8):825-30. -   2. Agresti A, Bianchi M E. HMGB proteins and gene expression. Curr     Opin Genet Dev. 2003; 13(2):170-8. -   3. Scaffidi P, Misteli T, Bianchi M E. Release of chromatin protein     HMGB1 by necrotic cells triggers inflammation. Nature. 2002;     418(6894):191-5. -   4, Ulloa L, Messmer D. High-mobility group box 1 (HMGB1) protein:     friend and foe. Cytokine Growth actor Rev. 2006; 17(3):189-201. -   5, Wang H, Vishnubhakat J M, Bloom O, Zhang M, Ombrellino M, Sama A,     et al. Proinflammatory cytokines (tumor necrosis factor and     interleukin 1) stimulate release of high mobility group protein-1 by     pituicytes. Surgery. 1999; 126(2):389-92. -   6. Messmer D, Yang H, Telusma O, Knoll F, Li J, Messmer B, et al.     High mobility group box protein 1: an endogenous signal for     dendritic cell maturation and Th1 polarization. J. Immunol. 2004;     173(1):307-13. -   7. Telusma G, Datta 5, Mihajlov I, Ma W, Li J, Yang H, et al.     Dendritic cell activating peptides induce distinct cytokine     profiles. Int Immunol. 2006; 18(11):1563-73. -   8. Saenz R, Souza Cda S, Huang C T, Larsson M, Esener S, Messmer D.     HMGB1-derived peptide acts as adjuvant inducing immune responses to     peptide and protein antigen. Vaccine. 2010; 28(47):7556-62. PMCID:     2963688. -   9. Yang H, Hreggvidsdottir H S, Palmblad K, Wang H, Ochani M, Li J,     et al. A critical cysteine is required for HMGB1 binding to     Toll-like receptor 4 and activation of macrophage cytokine release.     Proc Natl Acad Sci USA. 2010; 107(26):11942-7. PMCID: 2900689. -   10. Hoppe O, Talcott K E, Bhattacharya S K, Crabb J W, Sears J E.     Molecular basis for the redox control of nuclear transport of the     structural chromatin protein Hmgb1. Exp Cell Res. 2006;     312(18):3526-38. -   11. Bienkiewicz E A, Moon Woody A, Woody R W. Conformation of the     RNA polymerase II C-terminal domain: circular dichroism of long and     short fragments. J Mol. Biol. 2000; 297(0:119-33. -   12. van Holst G J, Fincher G B. Polyproline II Confirmation in the     Protein Component of Arabinogalactan-Protein from Lolium     multiflorum. Plant Physiol. 1984; 75(4):1163-4. PMCID: 1067069, -   13. Yu H, Chen J K, Feng S, Dalgarno D C, Brauer A W, Schreiber S L.     Structural basis for the binding of proline-rich peptides to SH3     domains. Cell. 1994; 76(5):933-45. -   14. Li S S. Specificity and versatility of SH3 and other     proline-recognition domains: structural basis and implications for     cellular signal transduction. Biochem J. 2005; 390(Pt 3):641-53.     PMCID: 1199657. -   15. Linardakis E, Bateman A, Phan V, Ahmed A, Gough M, Olivier K, et     al. Enhancing the efficacy of a weak allogeneic melanoma vaccine by     viral fusogenic membrane glycoprotein-mediated tumor cell-tumor cell     fusion. Cancer Res. 2002; 62(19):5495-504. -   16. Singh M, O_Hagan D. Advances in vaccine adjuvants. Nature     Biotechnology. 1999; 17(11):1075-81. -   17. McCluskie M J, Weeratna RD. Novel adjuvant systems. 2001;     1(3):263-71. -   18. Nunez Miguel R, Wong J, Westoll J F, Brooks H J, O'Neill L A,     Gay N J, et al. A dimer of the Toll-like receptor 4 cytoplasmic     domain provides a specific scaffold for the recruitment of     signalling adaptor proteins. PLoS One. 2007; 2(8):e788. PMCID:     1945083. -   19. Pujals S, Giralt E. Proline-rich, amphipathic cell-penetrating     peptides. Adv Drug Deliv Rev. 2008; 60(4-5):473-84. -   20. Fernandez-Cameado J, Kogan M J, Van Mau N, Pujals S,     Lopez-Iglesias C, Heitz F, et al. Fatty acyl moieties: improving     Pro-rich peptide uptake inside HeLa cells. J Pept Res. 2005;     65(6):580-90. 

1. An immunostimulatory peptide consisting of the amino acid sequence SAFFLFCSE.
 2. An immunostimulatory peptide comprising the amino acid sequence DPNAPKRPPSAFFLX₁X₂X₃X₄ or a derivative thereof, Wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; Wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; Wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or Wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.
 3. The immunostimulatory peptide of claim 2, wherein DPNAPKRPPSAFFLX₁X₂X₃X₄ has the amino acid sequence: a. DPNAPKRPPSAFFLX₁CSE, b. DPNAPKRPPSAFFLFX₁SE, c. DPNAPKRPPSAFFLFCX₁E, or Wherein X₁ is alanine (A), glycine (G), or valine (V).
 4. The immunostimulatory peptide of claim 2, wherein DPNAPKRPPSAFFLX₁X₂X₃X₄ is further mutated so that F at amino acid positions 12 and 13 is changed to S.
 5. The immunostimulatory peptide of claim 2, wherein derivative is a fragment of DPNAPKRPPSAFFLX₁X₂X₃X₄ having the sequence RPPSAFFLX₁X₂X₃X₄, Wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; Wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; Wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or Wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.
 6. The immunostimulatory peptide of claim 5, wherein RPPSAFFLX₁X₂X₃X₄ has the amino acid sequence: a. RPPSAFFLX₁CSE, b. RPPSAFFLFX₁SE, c. RPPSAFFLFCX₁E, or d. RPPSAFFLFCSX₁, Wherein X₁ is alanine (A), glycine (G), or valine (V).
 7. The immunostimulatory peptide of claim 6, wherein RPPSAFFLX₁X₂X₃X₄ is further mutated so that F at amino acid positions 6 and 7 is changed to S.
 8. The immunostimulatory peptide of claim 2, wherein the derivative is a fragment of DPNAPKRPPSAFFLX₁X₂X₃X₄ having the amino acid sequence SAFFLX₁X₂X₃X₄, Wherein when X1 is alanine (A), glycine (G), or valine (V) then X2 is C, X3 is S and X4 is E; Wherein when X2 is alanine (A), glycine (G), or valine (V) then X1 is F, X3 is S and X4 is E; Wherein when X3 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X4 is E; or Wherein when X4 is alanine (A), glycine (G), or valine (V) then X1 is F, X2 is C and X3 is S.
 9. The immunostimulatory peptide of claim 8, wherein SAFFLX₁X₂X₃X₄ has the amino acid sequence: a. SAFFLX₁CSE, b. SAFFLFX₁SE, c. SAFFLFCX₁E, or d. SAFFLFCSX₁, Wherein X₁ is alanine (A), glycine (G), or valine (V).
 10. The immunostimulatory peptide of claim 8, wherein SAFFLX₁X₂X₃X₄ is mutated so that F at amino acid positions 3 and 4 is changed to S. 11.-17. (canceled)
 18. A vaccine formulation comprising an antigen and an immunostimulatory peptide of claim 1 or
 2. 19. The vaccine formulation of claim 18, wherein the antigen is selected from a group consisting of an respiratory disease antigen, tumor related antigen, allergy related antigen, and cardiovascular disease antigen.
 20. The vaccine formulation of claim 19, wherein the respiratory disease antigen is a respiratory virus.
 21. The vaccine formulation of claim 20, wherein the respiratory virus is selected from a group consisting of a rhinovirus, influenza virus, coronavirus RSV, or a combination thereof.
 22. The vaccine formulation of claim 21, wherein the influenza virus is selected from the group consisting of H1N1, H1N2, H1N7, H2N2, H3N1, H3N2, H3N8, H4N8, H5N1 H5N2, H5N3, H5N8, H5N9, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, and any other subtypes arising from re-assortment between influenza A viruses. 23.-27. (canceled)
 28. A method of inducing CTL proliferation comprising contacting the vaccine of claim 18 with lymphocytes thereby inducing CTL proliferation.
 29. A method of enhancing an immune response in a subject by inducing CTL proliferation by the method of claim 28 thereby enhancing an immune response in a subject. 30.-31. (canceled)
 32. A method of enhancing an immune response in a subject comprising administering a sufficient amount of the immunostimulatory peptide of claim 1 or 2 thereby enhancing the immune response in the subject.
 33. A method for selectively eliciting a cell-mediated immune response but not a humoral immune response in a subject comprising administering a sufficient amount of the vaccine of claim 18 thereby enhancing the cell-mediated immune response in the subject.
 34. (canceled)
 35. A method of inhibiting tumor growth associated with a cancerous disease in a subject comprising administering to the subject the immunostimulatory peptide of claim 1 or 2, thereby inhibiting tumor growth in a subject. 36.-50. (canceled) 